Layered nanoparticles

ABSTRACT

The invention provides a process for forming a layered nanoparticle, comprising providing a suspension comprising a core particle in a first liquid, adding a second liquid to the suspension, and adding a reagent, or a precursor for the reagent, to the suspension. The second liquid is immiscible with the first liquid. If the reagent is added to the suspension, the reagent reacts to form a layer on the core particle to form the layered nanoparticle. If a precursor for the reagent is added to the suspension, the precursor is converted to the reagent, and the reagent reacts to form a layer on the core particle to form the layered nanoparticle.

This application is a divisional of U.S. application Ser. No.11/816,052, which was filed on Oct. 23, 2007, which in turn is anational stage entry of International Application No. PCT/AU2006/000193,which was filed on Feb. 14, 2006, which in turn claims priority toAustralian Application No. 2005900677, which was filed on Feb. 14, 2005,wherein the entireties of said patent applications are incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates to layered nanoparticles, and to processesfor making them.

BACKGROUND OF THE INVENTION

It is generally believed that controlled drug delivery is capable ofimproving the safety and clinical efficacy of cancer chemotherapeuticdrugs, which typically produce severe side effects due to non-specifictoxicity. One approach to this problem is to preferentially delivercytotoxic drugs to the tumour. It has been established by severalresearch groups that nanospherical particles in the 50-250 nm diameterrange, possessing the appropriate physico-chemical properties, can beselectively distributed into tumour masses from the general circulation,over a period of one to two days after intravenous injection. Thisoccurs by virtue of the aberrant structure of the micro-vasculaturewithin many tumours. Various types of nanoparticulate delivery systemshave been used experimentally but most have significant limitations thathave precluded their final use for medical applications.

The major limitations are as follows:

-   -   1) Their physical stability is too poor to provide long enough        blood circulation to ensure accumulation into the tumour (e.g.        liposomes),    -   2) The rate of release from most types of nano-particles is too        fast to provide a concentrated dose to the tumour,    -   3) The stable nano-particles developed to date have very low        drug loading and slow release rate which are insufficient to        deliver the appropriate therapeutic dose,    -   4) Many types of nanoparticulate systems are rapidly detected by        the immune system (i.e. reticulo-endothelial system) and        eliminated from the blood stream, resulting in a small        proportion of the drug reaching the tumour.

Most of the materials used as drug delivery vehicle are organic innature: polymers, liposomes, dendrimers, etc. In contrast, the ceramicmaterials provide many advantages over the organic delivery matrices.For example silica particles are biologically inert and have hydrophilicsurfaces. They are also non-toxic, highly biocompatible and can besynthesised at low temperature in order to preserve the molecularstructure of the drug. Furthermore, their size and porosity remainstable within a wide range of chemical environment. Sol-gel technology,an inorganic room temperature polymerisation technique (see FIG. 1), hasbeen used to successfully encapsulate organic molecules inside oxidematrices.

During the last several years, the present inventors have developed atechnology for producing ceramic particles for controlled releaseapplications (WO01/62332). This technology allows active molecules to beencapsulated in ceramic particles using a combination of sol-gelchemistry and water-in-oil (W/O) emulsion synthesis. The size of theparticle is controlled by the size of the emulsion droplets and therelease kinetics is controlled by the sol-gel chemistry. To producemonodisperse nanoparticles using this method, stable micro-emulsions areused. In such systems, the water droplet size is usually restricted toseveral to a few tens of nanometers limiting the final particle size toless than 100 nm even in the presence of important Ostwald ripening.Larger particle sizes can be obtained using unstable emulsion systemsbut the resulting particles present a broad size distribution which isundesirable in such application as for example the passive targeting oftumours where a precise control over the particle size is desirable.Although such a precise control over the size can be achieved using theStöber process (seeded growth in diluted media), this type of processdoes not provide the compartmentalisation achieved in emulsions which isnecessary to ensure encapsulation of the active materials duringgelation. Thus the Stöber process is inadequate for the synthesis ofparticles for controlled release applications. Another limitation of thetechnology outlined in patent WO01/62332 is its inability to produceparticles with delayed, sequential or pulsed release sequences. Once theceramic particles are introduced in a liquid, they start to releaseimmediately. This disadvantage might be overcome by producing acore-shell structure with the active molecule located in core surroundedby an empty shell which acts as a diffusion barrier and prevents theactive to leach rapidly.

Substantial work has been performed during the last decade to try toachieve complex and tailored release pattern of active molecules fromspecific matrices. The delayed-release, timed-release, or sequentialrelease of drug(s) from a variety of delivery vehicles have beeninvestigated. To achieve these complex release patterns, the deliverysystem is either based on the modification of physico-chemicalproperties of the delivery materials or the modification of themorphology of the system such as using multi-layered structure. Allthese systems use an organic matrix in various forms: polymer gel,liposome, fibre, microcapsule, tablet etc. Particles, and morespecifically nanoparticles, have not been investigated for these kindsof applications.

Core-shell colloidal materials with tailored structural, optical, andsurface properties have been intensely investigated over the lastdecade. The research in this area was driven by the potentialapplications of such colloids in a wide range of fields. Most of theresearch effort has concentrated on changing the surface properties of agiven particle by coating or encapsulating it within a shell of adifferent material. The core may be a metal oxide, a semiconductor, aquantum dot, a magnetic particle, a crystalline particles etc., whilethe shell usually changes the charge, the functionality, and thereactivity of the particle surface, and may also enhance the stabilityand dispersibility of the colloidal core. In other words, the materialof core is different from shell materials, and the most commonlyreported core-shell structures are ceramic core with polymer shell, orvice versa. Ceramic cores containing encapsulated actives and coatedwith a different kind of ceramic materials have also been reported inthe literature.

Several methods have been reported in the literature to grow ceramicparticles using sol-gel synthesis via W/O microemulsion. One such methoddepends on obtaining a larger particle size by adjusting the synthesisparameters. Although the particle size can be adjusted by controllingparameters such as the precursor concentration, water concentration, pH,temperature, ion strength, reaction time, there is a limitation on theparticle growth. It is difficult using this method to producemonodisperse particles larger than 100 nm due to the intrinsiccharacteristics of the reverse micelles. Another method reported in theliterature consist in extracting the particle seeds, drying them andthen redispersing these seeds in a fresh W/O microemulsion, followed bythe addition of more alkoxide precursors to grow the particles. Thereare two shortcomings regarding this method. First, during the extractionand drying steps the particles can aggregate irreversibly into micronsize agglomerates, and second, the procedure introduces an additionalseparation step to recover the solid particles from liquid, which cansignificantly decrease the overall yield. Yet another process that canbe used to increase particle size is described in FIG. 2. However, inthis case the active molecules can only be encapsulated in the core. Inaddition the particles growth is limited by the fact that the alkoxideprecursor needs to consume water for reaction. Since the amount of wateris dictated by the formation of W/O microemulsion, only a limited supplyof water is available. In fact, not all of the water introduced in thesystem is available for sol-gel reaction, as some water is bonded to thesurfactant. As more alkoxide is being added more water is being consumedand less is available for further hydrolysis and growth.

There is therefore a need for a layered nanoparticle in which a dopantis located in, and restricted to, the core of the nanoparticle and/orone or more of the layers surrounding the core, and for a process formaking such a nanoparticle. There is also a need for a process capableof making core-shell ceramic nanoparticles with more than oneencapsulated molecular species (dopant) or with the active (dopant)encapsulated in different discrete locations or layers (i.e. shells).Such a process may open up a wide range of novel potential applicationssuch as optical storage, data encryption or security ink in addition tothe controlled release of drugs described above.

OBJECT OF THE INVENTION

It is an object of the present invention to overcome or substantiallyameliorate at least one of the above disadvantages. It is a furtherobject to at least partially satisfy at least one of the above needs.

SUMMARY OF THE INVENTION

In a broad form of the invention there is provided a process for forminga layered nanoparticle, comprising exposing a catalyst to a reagent inthe presence of a core particle, whereby the reagent reacts to form alayer on the core particle to form the layered nanoparticle. Thecatalyst, reagent and core particle may be disposed in a fluid. Thefluid may be a liquid. The catalyst may be located on and/or in and/oraround the core particle. The catalyst may be a catalyst for reaction ofthe reagent. The catalyst may be located on the surface of the particle.The reaction may comprise one or more of hydrolysis, condensation,polycondensation, crosslinking, polymerisation, precipitation andgelation. There may be a dopant surrounding the core particle, and thereaction of the reagent may encapsulate the dopant in the layer. Thecore particle may form the core of the layered nanoparticle. Thus thelayered nanoparticle, once formed, may comprise the core particle atleast partially surrounded or encapsulated by the layer. The coreparticle may be completely surrounded or encapsulated by the layer. Thecore particle may be a single particle or an agglomerate of two or moreparticles. The core particle may comprise the same material as the layeror it may comprise a different material.

In one form the present invention provides a process for forming alayered nanoparticle, comprising:

-   -   providing a suspension comprising a core particle in a first        liquid; and    -   exposing a catalyst to a reagent in the presence of the core        particle,        whereby the reagent reacts to form a layer on the core particle        to form the layered nanoparticle.

The step of providing the core particle in the liquid may compriseforming the core particle in the first liquid. In an embodiment, theparticle is not separated from the first liquid before the step ofexposing. The first liquid may be non-polar.

The step of exposing may be repeated one or more times, thereby forminga plurality of layers. The step of exposing may comprise the steps of:

-   -   adding a second liquid to the suspension, said second liquid        comprising the catalyst; and    -   adding the reagent, or a precursor for the reagent, to the        suspension;        whereby, if the reagent is added to the suspension, the reagent        reacts to form a layer on the core particle, and if a precursor        for the reagent is added to the suspension, the precursor is        converted to the reagent, and the reagent to reacts to form a        layer on the core particle. The catalyst may be dissolved in the        second liquid. The second liquid may be immiscible with the        first liquid. The catalyst may be a catalyst for conversion of        the precursor to the reagent and/or for formation of the layer        from the reagent.

In another form the present invention provides a process for making alayered nanoparticle comprising:

-   -   providing a suspension comprising a core particle in a first        liquid;    -   adding a second liquid to the suspension, said second liquid        comprising a catalyst for reaction of a reagent; and    -   adding the reagent, or a precursor for the reagent, to the        suspension so as to cause the reagent to react to form a layer        on the core particle.

In another form the present invention provides a process for forming alayered nanoparticle, comprising:

-   -   providing a suspension comprising a core particle in a first        liquid;    -   adding a second liquid to the suspension, said second liquid        being immiscible with the first liquid; and    -   adding a reagent, or a precursor for the reagent, to the        suspension comprising the second liquid;        whereby, if the reagent is added to the suspension comprising        the second liquid, the reagent reacts to form a layer on the        core particle, and if a precursor for the reagent is added to        the suspension comprising the second liquid, the precursor is        converted to the reagent, and the reagent thus formed reacts to        form a layer on the core particle, to form the layered        nanoparticle. The second liquid may comprise a catalyst. If the        second liquid does not comprise a catalyst, the reagent may be        such as to be capable of forming the layer without a catalyst,        and/or the precursor may be capable of forming the reagent        without a catalyst. For example, if the second liquid is        aqueous, the precursor may be a silane that hydrolyses in the        absence of a catalyst.

In another form, the process comprises:

-   -   providing a suspension comprising a core particle in a first        liquid;    -   adding a second liquid to the suspension, said second liquid        comprising a catalyst for reaction of a reagent; and    -   adding a precursor for the reagent to the suspension so as to        cause the precursor to be converted to the reagent, and so as to        cause the reagent to react to form a layer on the core particle.

The second liquid may be immiscible with the first liquid. The step ofadding the second liquid may comprise depositing the second liquid onthe core particle, for example on the surface of the core particle. If aprecursor is added to the suspension, the precursor may be capable ofreacting with the second liquid to form the reagent, i.e. it may be aprecursor for the reagent. The reagent may be a condensable species, acrosslinkable species or a polymerisable species. The reaction of thereagent may form a solid layer or a gel layer on the core particle, andmay comprise condensation, crosslinking and/or polymerisation of thereagent. The core particle may be a nanoparticle and may be betweenabout 5 and 150 nm in diameter, or may be a submicron particle, and maybe less than about 500 nm in diameter. The core particle and/or thelayer may be solid, and may be porous, for example microporous and/ormesoporous. The second liquid may comprise a dopant, whereby theformation of the layer encapsulates the dopant in the layer. The step ofadding the second liquid and adding the reagent may be repeated one ormore times, thereby forming a plurality of layers. The core particle andeach layer independently may or may not have a dopant, and the dopants,if present, may be the same or different. The or each dopant may be areleasable substance, and may be releasable from the nanoparticle. Theprocess may generate a nanoparticulate substance comprising a pluralityof layered nanoparticles, whereby the step of providing a suspensioncomprises providing a suspension comprising a plurality of coreparticles in the first liquid. The core particles, and independently thelayered nanoparticles of the nanoparticulate substance, may behomogeneous in shape or may be heterogeneous in shape. They may bemonodispersed, or may have a narrow particle size distribution, or theymay have a broad particle size distribution.

In one aspect of the invention there is provided a process for making alayered nanoparticle comprising:

-   -   a) providing a suspension comprising a core particle, a first        surfactant and optionally a first cosurfactant in a first        non-polar liquid;    -   b) adding an aqueous liquid to the suspension, said aqueous        liquid comprising a catalyst for condensation of a condensable        species and optionally also comprising a dopant or a combination        of dopants;    -   c) adding a second non-polar liquid, a second surfactant and        optionally a second co-surfactant, said second non-polar liquid        being miscible with the first non-polar liquid; and    -   d) providing the condensable species (the reagent) to the        suspension such that the condensable species condenses in the        presence of the catalyst to form a layer at least partly        surrounding the core particle to generate the layered        nanoparticle.

The second non-polar liquid may be the same as or different to the firstnon-polar liquid. The second surfactant may be the same as or differentto the first surfactant. The second co-surfactant may be the same as ordifferent to the first co-surfactant. The second non-polar liquid,second surfactant and second co-surfactant may be added such that theratios of first non-polar liquid plus second non-polar liquid to firstsurfactant plus second surfactant and to first co-surfactant plus secondco-surfactant remain constant on a v/v. v/w, w/w, v/mol or w/mol basis.The suspension may be provided at a temperature of between about 1 and70° C., or the process may comprise bringing the suspension to atemperature between about 1 and 70° C., commonly between about 15 and40° C., and more commonly between 20 and 30° C., i.e. ambienttemperature. Lower temperatures may be used provided that thetemperature is such that neither the dispersed phase nor the continuousphase freezes. Higher temperatures may at times be used, depending onthe volatility and boiling point of the two phases and the inversiontemperature of the surfactant. Step d) may comprise providing ahydrolysable species to the suspension to form the layered nanoparticle,said hydrolysable species being capable of hydrolysing in the aqueousliquid to form the condensable species. Step d) may comprise the stepsof hydrolyzing the hydrolysable species in the aqueous liquid and ofcondensing the condensable species in the aqueous liquid to form thelayer. The process may comprise the step of maintaining the suspensionat a sufficient temperature (for example between about 1 and 70° C.) fora sufficient time (for example between about 6 and 96 hours) tohydrolyse the hydrolysable species, and optionally also to condense thecondensable species. Steps b) to d) may be repeated at least once, ormay be repeated 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times. Ineach repetition, the aqueous liquid may comprise a dopant or acombination of dopants, or may comprise no dopant, and the dopant in anyrepetition, if present, may be the same as or different to the dopant inany other repetition. Each repetition may form a layer, and each layermay comprise a dopant, or may comprise no dopant. The or each dopant maybe a releasable substance, or may be a non-releasable substance. Thecore particle may be between about 5 and 150 nm in diameter or may beless than 500 nm in diameter. The or each layer may have a meanthickness between about 1 and 100 nm, or between about 1 and 50 nm, 5and 75 nm or 5 and 30 nm. The process may be capable of controlling thethickness of the or each layer. The core particle may be porous, and maybe microporous and/or may be mesoporous and may have a pore size betweenabout 0.5 and 20 nm. The core particle may comprise a dopant, which maybe the same as or different to the dopant in the layer, or in any or allof the layers. The hydrolysable species in any repetition may be thesame as or different to the hydrolysable species in any otherrepetition. One or both of the suspension and the aqueous liquid maycomprise a surfactant. The process may generate a nanoparticulatesubstance comprising a plurality of layered nanoparticles, whereby thestep of providing a suspension comprises providing a suspensioncomprising a plurality of core particles in the first non-polar liquid.

The process may additionally comprise one or more of the followingsteps:

-   -   e) at least partially separating the layered nanoparticle, or        nanoparticulate substance, from the first non-polar liquid;    -   f) washing the layered nanoparticle, or nanoparticulate        substance, with an aqueous liquid;    -   g) washing the layered nanoparticle, or nanoparticulate        substance, with an organic liquid;    -   h) drying the layered nanoparticle, or nanoparticulate        substance.

Step f) may be conducted at room temperature, or may be conducted ashigh as 80° C. (e.g. at about 20, 30, 40, 50, 60, 70 or 80° C.). Theorganic liquid of step g) may be polar or non-polar. Its polarity may beselected depending on the solubility of dopants in the organic liquid.Step g) may be conducted at room temperature, or may be conducted ashigh as 70° C. (e.g. at about 20, 30, 40, 50, 60 or 70° C.), but shouldbe conducted at or below the boiling point of the organic liquid. Stepsf and g may be conducted more than once, and may be conducted in anyorder, or one or both may be omitted. For example, the process maycomprise (in that order) steps e and h, or steps e, f and h, or steps e,g and h, or steps e, f, g and h, or steps e, g, f and h, or steps e, g,f, g and h, or may comprise some other order of steps. Step h may beconducted in a manner that prevents or inhibits aggregation, for examplestep h may comprise freeze drying as described in W01/62332 (Barbé andBartlett, “Controlled Release Ceramic Particles, Compositions thereof,Processes of Preparation and Methods of Use”). Step h may be performeddirectly after step e. In that case, the large amount of surfactantpresent at that stage may prevent physical contact between the particlesand thus inhibit aggregation.

In an embodiment there is provided a process for making a layerednanoparticle having a dopant therein or thereon, comprising:

-   -   a) providing a stable suspension comprising a core particle, a        surfactant and optionally a cosurfactant in a non-polar liquid;    -   b) adding an aqueous liquid to the suspension, said aqueous        liquid comprising the dopant and a catalyst for condensation of        a condensable species;    -   c) adding a second non-polar liquid, a second surfactant and        optionally a second co-surfactant; and    -   d) adding a hydrolysable species to the suspension to form the        layered nanoparticle having the dopant therein or thereon, said        hydrolysable species being capable of hydrolysing in the aqueous        liquid to form the condensable species.

One or both of the suspension and the aqueous liquid may comprise asurfactant. The surfactant may be dissolved in the aqueous liquid or inthe suspension or in both.

In another embodiment the process comprises:

-   -   a) providing an emulsion comprising aqueous droplets dispersed        in a non-polar liquid, said emulsion additionally comprising a        surfactant and optionally a co-surfactant, wherein the droplets        comprise a catalyst for condensation of a first condensable        species upon hydrolysis;    -   b) adding a first hydrolysable species to the emulsion, said        first hydrolysable species being capable of hydrolyzing in the        aqueous droplets to form the first condensable species, thereby        forming a suspension of core particles in the non-polar liquid;    -   c) adding an aqueous liquid to the suspension, said aqueous        liquid comprising a catalyst for condensation of a second        condensable species;    -   d) adding a solution of a second surfactant, and optionally a        second cosurfactant, to the suspension; and    -   e) adding a second hydrolysable species to the suspension, said        second hydrolysable species being capable of hydrolysing in the        aqueous liquid to form the second condensable species.

The emulsion may be a water-in-oil (W/O) emulsion. Following theaddition of the first hydrolysable species (step b) or the secondhydrolysable species (step e), the hydrolysable species may hydrolyse toform the first or second condensable species respectively, which maythen condense due to the action of the catalyst(s) to form a layer atleast partially surrounding the core particles. The first hydrolysablespecies and the second hydrolysable species may be the same or they maybe different. Steps c) to e) may be repeated at least once, or may berepeated 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times, whereby eachrepetition forms a layer at least partially surrounding the coreparticles. In each repetition, the aqueous liquid may comprise a dopantor may comprise no dopant, and the dopant in any repetition may be thesame as or different to the dopant in any other repetition. The emulsionand/or the aqueous liquid may comprise a dopant, and at least one regionselected from the group consisting of the core particles and thelayer(s) may comprises at least one dopant.

In another embodiment there is provided a process for making layerednanoparticles comprising:

-   -   a) providing a surfactant solution comprising a surfactant and a        non-polar liquid, and optionally a co-surfactant;    -   b) adding to the surfactant solution an aqueous solution        comprising a catalyst for condensation of a first condensable        species and, optionally, also comprising a first dopant (core        dopant);    -   c) forming an emulsion from the surfactant solution and the        aqueous solution;    -   d) adding a first hydrolysable species to the emulsion, said        first hydrolysable species being capable of hydrolysing in the        aqueous solution to form the first condensable species;    -   e) maintaining the emulsion at a sufficient temperature for        sufficient time for formation of a suspension of core particles        from the first hydrolysable species;    -   f) adding an aqueous liquid to the suspension, said aqueous        liquid comprising a catalyst for condensation of a second        condensable species, and optionally a second dopant (layer        dopant);    -   g) adding a solution of a second surfactant, and optionally a        second cosurfactant, to the suspension; and    -   h) adding a second hydrolysable species to the suspension to        form the layered nanoparticles, wherein the second hydrolysable        species is capable hydrolysing in the aqueous liquid to form the        second condensable species.

Following the addition of the first hydrolysable species or the secondhydrolysable species, the hydrolysable species may hydrolyse to form thefirst or second condensable species respectively, which may thencondense due to the action of the catalyst(s).

The emulsion may be a microemulsion.

In another embodiment there is provided a process for making layerednanoparticles comprising:

-   -   a) providing a surfactant solution comprising a surfactant and a        non-polar liquid, and optionally a co-surfactant;    -   b) adding to the surfactant solution an aqueous solution        comprising a catalyst for condensation of a first condensable        species and, optionally, also comprising a first dopant;    -   c) forming an emulsion from the surfactant solution and the        aqueous solution;    -   d) adding a first hydrolysable species to the emulsion, said        first hydrolysable species being capable of hydrolysing in the        aqueous solution to form the first condensable species;    -   e) maintaining the emulsion at a sufficient temperature for        sufficient time for formation of a suspension of core particles        from the first hydrolysable species;    -   f) adding an aqueous liquid to the suspension, said aqueous        liquid comprising a catalyst for condensation of a second        condensable species, and optionally a second dopant;    -   g) adding a solution of a second surfactant, and optionally a        second cosurfactant, to the suspension;    -   h) adding a second hydrolysable species to the suspension to        form layered nanoparticles, wherein the second hydrolysable        species is capable hydrolysing in the aqueous liquid to form the        second condensable species;    -   i) at least partially separating the layered nanoparticles from        the non-polar liquid;    -   j) washing the layered nanoparticles with an organic liquid;    -   k) washing the layered nanoparticles with an aqueous liquid;    -   l) washing the layered nanoparticles with an organic liquid        (which may be the same as or different to the organic liquid of        step j; and    -   m) drying the layered nanoparticles.

In another aspect of the invention there is provided a layerednanoparticle comprising a core particle (i.e. core) and one or morelayers at least partially surrounding said core particle. The layerednanoparticle may be spherical. The core particle may be a solid, and maybe a porous solid, for example a microporous or mesoporous solid, andmay be spherical or non-spherical. The mean diameter of the core may bebetween about 5 and 500 nm. The or each layer may, independently, be asolid layer or a gel layer. At least one region of the layerednanoparticle selected from the group consisting of the core and the oneor more layers may comprise a dopant, or a plurality (for example 2, 3,4, 5 or more than 5) of dopants. Thus there may be a core dopant and/orone or more layer dopants. The or each dopant may be substantiallyhomogeneously distributed in the region that comprises said dopant. Thusfor example, if the nanoparticle comprises a core and a single layer,and the core comprises a core dopant and the layer comprises a layerdopant, then the core dopant may be substantially homogeneouslydistributed in the core, and the layer dopant may be substantiallyhomogeneously distributed in the layer. The layered nanoparticle may bea microporous or mesoporous ceramic nanoparticle. The core and the oneor more layers may each, independently, be microporous or mesoporous.The or each layer, and optionally the core particle, may, independently,comprise a hydrolysed silane, for example a hydrolysed alkoxysilane, andmay comprise silica and/or a polysilsesquioxane. The or each layer, andoptionally the core particle, may, independently, comprise a ceramic oran oxide, e.g. a metal oxide. If more than one of the core and the oneor more layers comprises a dopant, then the dopants in each of these maybe the same or they may be different. The or each dopant mayindependently be releasable or non-releasable. The layered nanoparticlemay be capable of sequential release and/or delayed release of one ormore of the releasable dopants. The layered nanoparticle may have a meandiameter between about 10 and 500 nm. The core and the or each layermay, independently, be microporous or mesoporous, and may have poresbetween about 0.5 and 10 nm. The or each layer may, independently, havea mean thickness between about 1 and 50 nm. The dopant may be aninorganic substance or an organic substance. It may be a salt, or a dye,a catalyst, an active material or active substance, e.g. a biologicallyactive substance (for example a protein, polysaccharide, enzyme, drug,peptide etc.), a magnetic substance, a radioactive species, aradioactive tracer or some other type of dopant.

In one embodiment there is provided a layered nanoparticle comprising aporous core particle comprising a first dopant and a porous layercomprising a second dopant at least partially surrounding said coreparticle. At least one of the first and second dopants may be releasablefrom the layered nanoparticle. Thus the nanoparticle may comprise aporous core particle comprising a first releasable dopant and a porouslayer comprising a second releasable dopant at least partiallysurrounding said core particle. The nanoparticle may be capable ofreleasing the second releasable dopant and the first releasable dopantsequentially.

In another embodiment there is provided a layered nanoparticlecomprising a porous core particle comprising a first releasable dopant,a first porous layer surrounding the core particle, said first layercomprising no releasable dopant, and a second porous layer comprising asecond releasable dopant at least partially surrounding the first porouslayer. The nanoparticle may be capable of releasing the secondreleasable dopant and the first releasable dopant sequentially, with adelay between release of the second and first dopants. The length of thedelay may be dependent on the thickness and porosity of the first layer.

In another embodiment there is provided a layered nanoparticlecomprising a core particle comprising no releasable dopant and a porouslayer comprising a releasable dopant at least partially surrounding saidcore particle. The core particle may comprise a non-releasable dopant,for example a non-releasable pigment for colouring the nanoparticle. Thecore may be porous or non-porous. The core may comprise a magneticmaterial, so that the layered nanoparticle is magnetic.

In another embodiment there is provided a layered nanoparticlecomprising a non-porous core particle comprising, optionally consistingof, a releasable dopant and a porous layer comprising no releasabledopant, said porous layer at least partially surrounding the coreparticle. The nanoparticle may be capable of releasing the dopant aftera delay. The length of the delay may be dependent on the thickness andporosity of the porous layer. The layer may have a non-releasabledopant, for example a non-releasable pigment for colouring thenanoparticle.

There is also provided a nanoparticulate substance comprising aplurality of layered nanoparticles according to the invention. Thenanoparticulate substance may be monodispersed, or may have a narrowparticle size distribution.

There is also provided a layered nanoparticle when made by the processof the invention. Also there is provided a nanoparticulate substancecomprising a plurality of layered nanoparticles according to theinvention or when made by the process of the invention.

In another aspect of the invention there is provided a method fordelivering a releasable substance to a fluid comprising exposing alayered nanoparticle according to the invention, or a plurality oflayered nanoparticles according to the invention, to the fluid, whereinthe layered nanoparticle(s) has a releasable substance therein and/orthereon (e.g. in at least one of the layers or the core particle), andthe fluid is capable of at least partially releasing the releasablesubstance from the layered nanoparticle(s).

The fluid may be an aqueous fluid, for example a biological fluid, ormay be a non-aqueous fluid, for example an organic solvent. The exposingmay comprise stirring, shaking, swirling, sonicating or otherwiseagitating the layered nanoparticle(s) with the fluid. The method maycomprise releasing the releasable substance into the fluid, and maycomprise releasing the releasable substance in a controlled manner. Therelease of the releasable substance may be controlled variable release,whereby the rate of release of the releasable substance is variable withtime in a controlled and/or predetermined manner.

In an embodiment the releasable substance is a drug, and the fluid is abodily fluid e.g. blood in a patient. This embodiment comprises a methodfor administering the drug to the patient, said method comprisingdelivering to the patient, e.g. to the blood of the patient, a layerednanoparticle according to the invention, or a plurality of layerednanoparticles according to the invention, said layered nanoparticle(s)having the drug therein and/or thereon. The drug may be for example ananticancer drug. The layered nanoparticle(s) may have a diameter betweenabout 10 and 500 nm, or between about 50 and 300 nm. The delivery may beintravenous (IV) delivery, and may comprise injecting into the patient asuspension comprising the nanoparticle(s). The suspension may comprise asuspending fluid. Suitable suspending fluids for injections are wellknown, and may comprise saline, Ringer's solution, glucose solution,fructose solution, dextrose solution, amino acid solution, proteinhydrolysate, sodium lactate solution or some other aqueous liquid. Thereis also provided the use of a layered nanoparticle according to theinvention for administering a drug to a patient, said layerednanoparticle having the drug therein and/or thereon.

In another aspect of the invention there is provided a layerednanoparticle according to the invention, or a plurality of layerednanoparticles according to the invention, when used for delivering areleasable substance to a fluid.

The invention also provides for use of a layered nanoparticle accordingto the invention for selective sorption of at least one component of amixture. The invention further provides for a layered nanoparticle whenused for selective sorption of at least one component of a mixture.

BRIEF DESCRIPTION OF THE DRAWINGS

A preferred form of the present invention will now be described by wayof example with reference to the accompanying drawings wherein:

FIG. 1 shows a sol-gel reaction sequence in the presence of base;

FIG. 2 shows a flow chart of a process for producing multilayerednanoparticles with dopants contained in the core only;

FIG. 3 shows a flow chart of the process for producing multilayerednanoparticles with multiple dopants;

FIG. 4 shows TEM micrographs of particles produced in aNP-9/1-pentanol/cyclohexane/NH₄OH/TEOS system (average size 55±5 nm);

FIG. 5 shows TEM images of uniform silica particles with a core-shellstructure;

FIG. 6 shows a graph of TGA/DTA measurement of surfactant residue onsilica particles;

FIG. 7-a shows a TEM micrograph of silica particles produced in themicroemulsion system a) NP-5/cyclohexane/NH4OH/TMOS;

FIG. 7-b shows a TEM micrograph of silica particles produced in themicroemulsion system b) NP-9/1-pentanol/cyclohexane/NH4OH/TMOS:

FIG. 7-c shows a TEM micrograph of silica particles produced in themicroemulsion system c) Triton X-100/toluene/NH4OH/TMOS:

FIG. 8 shows TEM micrographs illustrating particle growth by sequentialaddition of TMOS: a) core (22 nm), b) 1st addition of 1.2 mmol of TMOS(27 nm), c) 2nd addition (31 nm), d) 3rd addition (33 nm), e) 4thaddition (36 nm);

FIG. 9 shows TEM micrographs illustrating particle growth by sequentialaddition of TEOS: a) core (50 nm), b) 1st addition of 1.2 mmol of TMOS(83-92 nm), c) 2nd addition (91-106 nm), d) 3rd addition (102-120 nm),e) 4th addition (108-129 nm), f) 5th addition (128-146 nm);

FIG. 10 shows a schematic diagram illustrating the principle of thenovel growth process;

FIG. 11-a shows a TEM micrograph of core-shell structure having: a) abright field image (dark areas indicate heavier elements);

FIG. 11-b shows a TEM micrograph of core-shell structure having: b) adark field image (bright areas indicate heavier elements);

FIG. 12-a shows a TEM micrograph of core-shell structure showing beamdamage having: a) a bright field image,

FIG. 12-b shows a TEM micrographs of core-shell structure showing beamdamage having: b) a dark field image after EDX line profile;

FIG. 13 shows micrographs illustrating the influence of the method ofaddition on the core-shell particle size.

FIG. 14 shows TEM micrographs of particles synthesised using: a) 4 cycle(core+4 shells), and b) 5 cycle (core+5 shells);

FIG. 15-a shows TEM micrographs of particles grown using differentdilution ratios, where the dilution rates (volume of freshemulsion/volume of seed emulsion) were: A) 2, B) 3, C) 4, D) 5 forsample 1, silica core copper doped, the extra added aqueous phase 1.333mol/L NH4OH, with Cu 10.14 mg/mL in the form of Cu(NH3)42+;

FIG. 15-b shows TEM micrographs of particles grown using differentdilution ratios, where the dilution rates (volume of freshemulsion/volume of seed emulsion) were: A) 2, B) 3, C) 4, D) 5 forsample 2, silica core copper doped, the extra added aqueous phase 1.333mol/L NH4OH without copper;

FIG. 16 shows TEM micrographs of particles prepared using differentmethods for mixing the seed emulsion and the fresh emulsion;

FIG. 17 shows TEM micrographs of particle cores formed using TEOS, andshells using TMOS (synthesis conditions identical to those used for FIG.5);

FIG. 18 shows TEM micrographs of silica core coated with Ormosil shell;

FIG. 19 shows TEM micrographs of samples with composition listed inTable 2;

FIG. 20 shows photographs of samples with composition listed in Table 1and suspended in acetone;

FIG. 21 shows TEM micrographs of samples with composition listed inTable 3;

FIG. 22 shows photographs of sample with composition listed in Table 3and suspended in acetone;

FIG. 23-a shows photographs of samples with composition listed in Table4 that is a) freeze-dried (SiO2/NaCl weight ratio: 15%);

FIG. 23-b shows photographs of samples with composition listed in Table4 that is b) suspended in Milli-Q water;

FIG. 24 shows photomicrographs of In₂O₃ nanocrystals in suspension andcoated with silica shell;

FIG. 25 shows UV-visible spectra of CuPC (black line, λ_(max): 556 nm)and Rubpy (grey line, λ_(max): 390 nm) in neutral water;

FIG. 26 shows UV-visible spectra of samples LNK-822 (black line,λ_(max): 615 nm) and LNK-824 (grey line, λ_(max): 450 nm) in SBF after32 days;

FIG. 27-a shows a release rate curve of a) Sample LNK-822 (1.022 g offreeze dried powder in 20 mL SBF at 37° C., max=615 nm;

FIG. 27-b shows a release rate curve of b) Sample LNK-824 (0.6055 g offreeze-dried powder in 20 mL SBF at 37° C., max=450 nm), wherein thefreeze dried powders contain 15 wt % of SiO2 and 85 wt % of NaCl;

FIG. 28a shows a diagrammatic representation of an embodiment of layerednanoparticles according to the present invention;

FIG. 28b shows a diagrammatic representation of an embodiment of layerednanoparticles according to the present invention;

FIG. 28c shows a diagrammatic representation of an embodiment of layerednanoparticles according to the present invention;

FIG. 28d shows a diagrammatic representation of an embodiment of layerednanoparticles according to the present invention;

FIG. 28e shows a diagrammatic representation of an embodiment of layerednanoparticles according to the present invention;

FIG. 28f shows a diagrammatic representation of an embodiment of layerednanoparticles according to the present invention;

FIG. 29 shows a release rate curve, measured using UV/visibleabsorption, of samples (A) LNK-879 and (B) LNK-880. 2 g of freeze driedpowder suspended in 20 mL SBF, at 37° C. (wherein the freeze driedpowders contain 15 wt % of SiO₂ and 85 wt % of NaCl): ▴ CuPC,λ_(max)=615 nm, ▪ Rubpy, λ_(max)=450 nm;

FIG. 30 shows a flow chart of a synthesis of dye encapsulated silicananoparticles with ORMOSIL surface by adding more emulsion components;

FIG. 31 shows TEM images of silica nanoparticles with ORMOSIL surfaceproduced by adding more emulsion components: (a) seeded by TEOS; (b)seeded by mixed precursors; (c) core-shell particles by TEOS; (d) and(e) silica core with ORMOSIL shell, (f) ORMOSIL core and shell, asdetailed in Table 6;

FIG. 32 shows TEM images of silica particles at various surfactantconcentrations: (a, b) seed particles made by 0.2 mol/L NP-9 and 0.2mol/L 1-pentanol, 74-86 nm; (c) core+2-layers particles by 0.4 mol/LNP-9 without 1-pentanol, 109-123 nm; (d) core+3-layers particles by 0.4mol/L NP-9 without 1-pentanol, 132-152+20 nm; (e) core+2-layersparticles by 0.6 mol/L NP-9 without 1-pentanol, 106-126 nm; (f)core+3-layers particles by 0.6 mol/L NP-9 without 1-pentanol, 132-145+20nm; (g) core+2-layers particles by 0.4 mol/L NP-9, 0.4 mol/L 1-pentanol,100-131 nm; (h) core+3-layers particles by 0.4 mol/L NP-9, 0.4 mol/L1-pentanol, 128-179 nm;

FIG. 33 shows a flow chart of production of layered silica nanoparticlesby combining two growth techniques according to the present invention;

FIG. 34 shows TEM images of silica particles produced by combining twogrowth techniques at mole ratio [H₂O]/[surfactant]=6: (a) Seed: 50-60nm, TEOS 1.2 mmol, H₂O 36 mmol, [H₂O]/[TEOS]=30; (b) Core+1-layer: 61-76nm, TEOS 4.8 mmol, H₂O 36 mmol, [H₂O]/[TEOS]=7.5; (c) Core+2-layer:95-113 nm, TEOS 9.6 mmol, H₂O 72 mmol, [H₂O]/[TEOS]=7.5; (d)Core+3-layer: 100-135 nm, TEOS 14.4 mmol, H₂O 72 mmol, [H₂O]/[TEOS]=5;(e) Core+4-layer: 136-166 nm, TEOS 24 mmol, H₂O 144 mmol,[H₂O]/[TEOS]=6; (f) Core+5-layer: 146-172 nm, TEOS 33.6 mmol, H₂O 144mmol, [H₂O]/[TEOS]=4.3; (g) Core+7-layer: 200-240 nm, TEOS 52.8 mmol,H₂O 216 mmol, [H₂O]/[TEOS]=4.1;

FIG. 35 shows TEM images of silica particles produced by combining twogrowth techniques at mole ratio [H₂O]/[surfactant]=9: (a) Seed: 50-61nm, TEOS 1.2 mmol, H₂O 54 mmol, [H₂O]/[TEOS]=45; (b) Core+1-layer: 70-87nm, TEOS 4.8 mmol, H₂O 54 mmol, [H₂O]/[TEOS]=11.3; (c) Core+2-layer:95-117 nm, TEOS 9.6 mmol, H₂O 162 mmol, [H₂O]/[TEOS]=16.9; (d)Core+3-layer: 158-172 nm, TEOS 14.4 mmol, H₂O 162 mmol,[H₂O]/[TEOS]=11.3; (e) Core+4-layer: two size nanoparticles, TEOS 24mmol, H₂O 378 mmol, [H₂O]/[TEOS]=15.8; (f) Core+5-layer: broad sizenanoparticles, TEOS 33.6 mmol, H₂O 378 mmol, [H₂O]/[TEOS]=11.3;

FIG. 36 shows TEM images of silica nanoparticles synthesised at hightemperatures with mole ratio of [H₂O]/[surfactant]=6: (a) 35-50 nmageing 20 hrs at 35±2° C.; (b) 48-62 nm ageing 48 hrs at 35±2° C.; (c)37-52 nm ageing 20 hrs at 50±2° C.; (d) 48-70 nm ageing 48 hrs at 50±2°C.;

FIG. 37 shows TEM images of silica nanoparticles synthesised at hightemperatures with mole ratio of [H₂O]/[surfactant]=8: (a) Seed: 35-68 nmat room temperature 35±2° C. ageing 48 hrs: Core-shell particles; (b)75-100 nm, ageing 20 hrs at 35±2° C.; (c) 78-113 nm, ageing 28 hrs at35±2° C.; (d) 81-117 nm, ageing 48 hrs at 35±2° C.; (e) 78-105 nm,ageing 20 hrs at 50±2° C.; (f) 80-107 nm, ageing 28 hrs at 50±2° C.; (g)86-120 nm, ageing 48 hrs at 50±2° C.; and

FIG. 38 shows TEM images of silica particles at different incubationstages: (a) before incubation (76-88 nm); (b) 55±5° C. incubating 1 hourbefore adding extra water (82-96 nm); (c) 55±5° C. incubating 6 hoursafter adding extra water (74-100 nm).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to inorganic/inorganic particles coreshell particles and, in some embodiments, to particles made out ofceramic materials, with a ceramic core and one or more ceramic layer, orshell. The invention is applicable to a variety of porous metal oxideshells and non-porous or porous (oxide or non oxide) cores for examplesilica.

Silica core-shell particles can be divided into:

-   -   Silica particles with incorporated dyes    -   Silica coated metal clusters    -   Core-shell organosilicon with incorporated dyes

The first and third categories are of particular interest for theencapsulation and release from core-shell particles. The particles ofthe present invention differ from the prior art in that a dopant may bedoped in any of the layers. Thus in many of their embodiments theparticles of the present invention may be considered as beingmulti-layered nanoparticles with at least one of the shell and a layercontaining at one or more dopants being homogeneously distributedthroughout the layer and capable of being released.

To be able to tailor the composition of each of the individual layers ofthe multi-layer particles it is possible to exploit the ability tochange the micelle pool compositions. This may be achieved, as describedherein, by sequential addition of the surfactant/water and a reagentprecursor. It may be important to maintain the emulsion composition(i.e. the same spot in the ternary phase diagram: Surfactant-Oil-Water)that allows production of homogeneous multi-layered particles withidentical core-shell structures and does not lead to generation ofparticles with different composition. Thus the present inventionprovides a cyclic addition of emulsion and precursor that facilitatesproduction of multilayered particles.

The present specification discloses a sol-gel process for producinglayered nanoparticles via a water-in-oil microemulsion (W/O) system. Thenanoparticles may be multi-layered. The resulting nanoparticles maycontain one or more active molecules, encapsulants and/or dopants,encapsulated in different locations within the nanoparticles, which maybe released in a controlled fashion. The nanoparticles may be ceramicnanoparticles. They may be synthesised by sequentially adding(different) active molecules (dopants) and (different) precursors (ororganically modified precursors), as well as fresh W/O emulsions. Theoverall particle size as well as the core diameter and each layerthickness may be tailored by controlling the amount of precursor added.The thickness of each layer may be controlled between about 1 and 50 nm.The precursor may be a hydrolysable species, e.g. a hydrolysable silane.It may be capable of hydrolyzing to form the reagent. The precursor maybe varied from silicon alkoxide to other metal alkoxide or a mixture ofmetal alkoxides. Depending on their location in the multilayeredparticles, the active molecules may be released sequentially or insegmented fashion (e.g. release-no release-release). Thus for example ifa nanoparticle is constructed having a releasable substance in the coreand in an outer layer, with an inner layer between the outer layer andthe core having no releasable substance, then release of the releasablesubstance would proceed stepwise, with release-no release-release stepsoccurring sequentially.

In the process of the present invention, the core particles may beformed in situ or they may be added as preformed particles. The coreparticles may be any species of powder, for example silica particles(such as fumed silica, colloidal silica or silica fume), metal oxideparticles (such as indium oxide), mixed metal oxide particles (such asindium oxide doped silica particles), semiconductor particles, quantumdots, magnetic particles, crystalline particles or some other type ofparticles. The core particles may be spherical, or cubic, or may be inthe shape of a triangular prism, a tetrahedron, a polyhedron (forexample with 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or20 or more than 20 faces), a flake or an irregular shape. The coreparticles may be crystalline or non-crystalline, or may be partlycrystalline. It may be porous or non-porous. The core particles may bebetween about 5 and 500 nm in diameter, or between about 5 and 450, 5and 400, 5 and 300, 5 and 200, 5 and 100, 5 and 50, 5 and 20, 10 and500, 10 and 400, 10 and 200, 10 and 100, 50 and 500, 50 and 400, 50 and200, 50 and 100, 100 and 500, 100 and 400, 100 and 200, 200 and 500 or300 and 500 nm, for example about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 nm indiameter, or some other diameter. The non-polar liquid (or eachnon-polar liquid independently if two or more are used in differentsteps) may be a hydrocarbon, and may have between 5 and 16 carbon atoms,or between 5 and 12, 5 and 8, 6 and 12 or 6 and 10, and may have 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon atoms. It may be straightchain, branched, cyclic and may be aliphatic or aromatic. It may be amixture of hydrocarbons. Alternatively it may be some other non-polarliquid, for example a haloalkane or haloaromatic liquid or a lowviscosity silicone, for example a low viscosity dimethylsilicone or alow viscosity fluorosilicone. The low viscosity silicone may be a linearsilicone or a cyclic silicone. The viscosity of the silicone may bebelow about 100 cS, or below about 50, 20, 10, 5, 2 or 1 cS, and may beabout 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 cS.The linear silicone may be a trimethylsiloxy ended silicone. The cyclicsilicone may be a D3, D4, D5, D6, D7 or D8 cyclic silicone or may be amixture of any two or more of these. A typical hydrocarbon that may beused is cyclohexane. The non-polar liquid may be recyclable. Thecatalyst may be a catalyst for the sol-gel process. It may be a catalystfor condensation of the condensable species, and may be for example astrong acid (such as sulfuric acid, hydrochloric acid), an organic acid(such as acetic acid, trifluoroacetic acid), a base (for example ahydroxide such as KOH, NaOH, aqueous ammonia), an amine (for exampleORMOSIL functionalized with amine such as APTES(aminopropyltriethoxysilane)), a fluoride (for example HF, NaF, KF,NH₄F) or a transition metal alkoxide (for example titanium alkoxide,vanadium alkoxide). The concentration of the catalyst in the aqueousliquid may be between about 0.1 and 5M and may be between about 0.1 and2, 0.1 and 1, 0.1 and 0.5, 0.5 and 5, 1 and 5, 3 and 5, 0.5 and 2 or 1and 2M, and may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5M. The pH of theaqueous liquid may be between about 8 and 14, or between about 8 and 13,8 and 12, 8 and 11, 8 and 10, 9 and 13, 10 and 13, 11 and 13, 9 and 12,10 and 12 or 11 and 12, and may be about 8, 8.5, 9, 9.5, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5 or 14. The ratio of the aqueous liquid to thesuspension of core particles on a v/v basis may be between about 0.1 and10%, or between about 0.1 and 5, 0.1 and 2, 0.1 and 1, 0.5 and 10, 1 and10, 5 and 10, 0.5 and 5, 0.5 and 2 or 1 and 2%, and may be about 0.1,0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5 or 10%. The ratio of the hydrolysable species to the aqueous liquidmay be between about 0.5 and 10% on a w/w, w/v, v/v or molar basis, andmay be between about 0.5 and 5, 0.5, and 2, 0.5 and 1, 1 and 10, 5 and10 or 2 and 5%, and may be about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10% on a w/w, w/v, v/v or molarbasis. The hydrolysable species may be a hydrolysable metal species, forexample a hydrolysable silicon, aluminium or titanium species. Thehydrolysable species may be an alkoxide, such as a metal alkoxide (e.g.silicon alkoxide, titanium alkoxide or aluminium alkoxide). The alkoxidemay be a C1 to C6 straight chain or branched chain alkoxide or may be amixture of such alkoxides. Alternatively the hydrolysable species may bean aryloxide, for example phenoxide, a silicon alkanoate, for example asilicon propionate, and aminosilane, an amidosilane or some otherhydrolysable silane. The metal may have between 2 and 4 hydrolysablegroups per molecule on average, and may have about 2, 2.5, 3, 3.5 or 4hydrolysable groups per molecule on average. Suitable hydrolysablespecies include, but are not restricted to, tri- and tetra-alkoxysilanessuch as tetramethoxysilane (TMOS), tetraethoxysilane (TEOS),tetrabutoxysilane (TBOS), tetrapropoxysilane (TPOS),methyltrimethoxysilane (MTMS), methyltriethoxysilane (MTES),ethyltriethoxysilane (ETES), ethyltrimethoxysilane (ETMS),octyltriethoxysilane (OTES), octyltrimethoxysilane (OTMS),hexadecyltrimethoxysilane (HDTMS), hexadecyltriethoxysilane (HDTES),octadecyltrimethoxysilane (ODTMS), octadecyltriethoxysilane (ODTES), aswell as methyl polysilicate (MPS), ethyl polysilicate (EPS),polydiethoxysilane (PDES), hexamethyl disilicate, hexaethyl disilicateor functional trialkoxysilanes such asmethacryloyloxypropyltrimethoxysilane, phenyltriethoxysilane (PTES),phenyltrimethoxysilane (PTMS), glycidoxypropyltrimethoxysilane (GLYMO),glycidoxypropyltriethoxysilane (GLYEO), mercaptopropyltriethoxysilane(MPTES), mercaptopropyltrimethoxysilane (MPTMS),aminopropyltrimethoxysilane (APTMS), aminopropyltriethoxysilane (APTES),3-(2-aminoethylamino)propyltrimethoxysilane (DATMS),3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane (TATMS),[2-(cyclohexenyl)ethyl]triethoxysilane (CHEETES), vinyltrimethoxysilane(VTMS), vinyltriethoxysilane (VTES) and mixtures of these. Thehydrolysable species may be capable of hydrolysing to produce acondensable species. The condensable species may be a partial orcomplete hydrolysate of any of the above hydrolysable species. It may bea silanol species, and may have 1, 2, 3 or 4 silanol groups permolecule, or more than 4 silanol groups per molecule. It may be an atleast partially condensed material having 1 or more silanol groups permolecule. It may be a mixture of silanol species. Thus the core and theone or more layers may, independently, comprise silica,polysilsesquioxane, alumina, titania or some other metal oxide. If thehydrolysable species is organofunctional, this may provide a condensablespecies which is similarly organofunctional, and ultimately may lead toa nanoparticle which is similarly organofunctional, optionallyselectively organofunctional in one or more of the core and thelayer(s). This may provide selective affinity for a dopant or otherspecies for the organofunctional core and/or layer(s). Alternatively, itmay be possible to provide a particle with core having a precursor to adesired compound, and one or more layers surrounding the core having acatalyst and/or reagent(s) for converting the precursor into the desiredcompound. In use, the precursor may be released from the core, and beconverted into the desired compound by the catalyst and/or reagent(s)for release from the particle. This may be of particular use if thedesired compound is of limited stability, and/or if the precursor ismore stable than the desired compound.

The sufficient temperature to hydrolyse may be between about 1 and 70°C., or between about 1 and 50, 1 and 30, 1 and 20, 1 and 10, 1 and 5, 10and 50, 10 and 40, 10 and 30, 10 and 20, 15 and 40, 20 and 50, 50 and70, 30 and 50, 20 and 40 or 20 and 30° C., and may be about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70° C.The sufficient time may be between about 6 and 96 hours, or betweenabout 6 and 72, 6 and 48, 6 and 24, 12 and 96, 24 and 96, 36 and 96, 48and 96, 60 and 96, 12 and 72, 24 and 60, 36 and 60 or 36 and 48 hours,and may be about 6, 12, 18, 24, 36, 48, 60, 72, 84 or 96 hours, or maybe more than 96 hours. The core particles may be provided at atemperature between about 1 and 70° C., or may be brought to atemperature between about 1 and 70° C., or between about 10 and 70° C.or between about 1 and 10° C. or between about 1 and 50° C. or betweenabout 1 and 20° C. or between about 10 and 50° C., or between about 10and 40, 10 and 30, 10 and 20, 20 and 50, 50 and 70, 30 and 60, 30 and50, 20 and 40 or 20 and 30° C., and may be about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70° C.

The dopant may be a salt (e.g. comprising a metal complex ion such ascopper tetramine), a dye, a catalyst, a biologically active substance(for example a protein, polysaccharide, enzyme, drug, peptide etc.), amagnetic substance, a radioactive tracer or other radioactive species,or some other type of dopant. There may be different dopants indifferent locations within the nanoparticles (e.g. the core and each ofthe one or more layers may comprise different dopants). The same dopantmay be located in different locations within the nanoparticles (e.g. thecore and one of the one or more layers may comprise the same dopant).The or each dopant may be releasable or non-releasable. If more than onedopant is present, at least one dopant may be releasable and at leastone dopant may be non-releasable, or all dopants may be releasable orall dopants may be non-releasable. A dopant in the core or a layer maybe distributed through said core or layer, and may be distributed evenlyor unevenly through said core or layer. It will be understood that wherereference is made to a dopant, this may include a plurality of dopants(for example 2, 3, 4, 5 or more than 5 dopants). If the core particle orany of the layers comprises a plurality of dopants, they may be allreleasable, or at least one may be releasable and at least onenon-releasable, or they may be all non-releasable. For example a layermay comprise a mixture of releasable dopants to be releasedsimultaneously, or it may comprise a mixture of non-releasable dopants(e.g. a dye and a magnetic dopant) to impart either a plurality ofproperties to the layer or a composite property (as for example if twodifferent dyes were incorporated into a single layer), or it maycomprise a releasable dopant for release to the environment surroundingthe particle, together with a non-releasable dopant (for example a dye).

The dopant in the core particle, if present, may be substantiallyhomogeneously distributed through the core particle. The dopant in thelayer, or any of the layers (if more than one layer is present) may besubstantially homogeneously distributed through that layer.

In an example, the layered nanoparticle may comprise a core particlehaving a non-releasable dye (e.g. red), and the core particle may besurrounded by a porous layer comprising a releasable dye of a differentcolour (e.g. green). Thus the layered nanoparticle may adopt the colourof the releasable dye (e.g. green), or a composite colour of thereleasable and non-releasable dyes (e.g. brown). When the layerednanoparticle adopts the colour of the non-releasable dye (e.g. red),then a user may be alerted to the fact that the releasable dye has beenreleased. In a particular example, the porous layer comprises a secondreleasable dopant (e.g. a reagent for a chemical reaction) in additionto the releasable dye. When the layered nanoparticle adopts the colourof the non-releasable dye, a user may be alerted to the fact that it isnecessary to add further nanoparticles in order to ensure continuedrelease of the releasable dopant.

The core particle may, for example, comprise a quantum dot, which mayfunction as a non-releasable dye. The core particle may be any suitableparticle, for example a porous or non-porous particle. It may be ananoparticle.

The dopant may comprise an immobilised reagent or catalyst. For example,a nanoparticle may comprise a core particle and layer surrounding thecore particle. The core particle may comprise a dopant (e.g. a prodrug)which is a precursor to a compound (e.g. a drug) to be released to theenvironment of the nanoparticle, and the layer may comprise a catalystfor converting the dopant into the compound. When the nanoparticle isexposed to a suitable environment, the dopant may pass from the coreparticle through the layer, where it would be converted under theinfluence of the catalyst into the compound, which would then bereleased from the nanoparticle. This may be useful in the case of acompound which is of limited long term stability.

A layer may comprise a release rate modifier, either in the form of anon-releasable dopant or in the form of the material of the layeritself. The release rate modifier may be capable of modifying therelease rate of a releasable dopant incorporated in the layer or in alayer closer to the core particle or in the core particle. The releaserate modifier may be capable of accelerating or decelerating the releaseof a releasable dopant. For example the core may comprise a releasabledopant which has acidic groups. A layer surrounding the core maycomprise amine groups, which would slow the release of the releasabledopant.

The core or one or more layers may comprise a sorbent, either in theform of a non-releasable dopant or in the form of the material of thelayer itself. For example, the core particle or a layer may comprise anon-releasable amine dopant, or the material of the core particle orlayer may comprise amine groups (for example derived fromaminopropyltriethoxysilane used in making the core particle or thelayer), for sorbing acidic species. An example of the use of such amaterial may be the selective sorption of low molecular acids in thepresence of high molecular weight acids. Thus a porous core particlehaving amine groups, for example derived fromaminopropyltrimethoxysilane, aminopropyltriethoxysilane oraminoethylaminopropyltrimethoxy- or trimethoxy-silane, may have aneutral porous layer applied to it to form a nanoparticle according tothe present invention. On exposure to a liquid comprising low and highmolecular weight acids, the low molecular weight acids would be capableof penetrating the layer to be sorbed into the core particle, whereashigher molecular weight acids may be prevented from accessing the coreparticle. The high molecular weight acids would be shielded from theamine groups in the core particle, thus preventing coagulation of theliquid due to an interaction between the nanoparticles and the highermolecular weight acids. In that manner, the low molecular weight acidsmay be selectively removed from the mixture without coagulation of theliquid. Another example of the use of such a material may be thesorption of highly toxic materials. Thus, for example, the nanoparticlehaving a porous core particle with amine groups surrounded by a neutralporous layer may be used for sorbing highly toxic acidic materialssafely. On exposure to a liquid comprising the toxic acidic material,the nanoparticle may sorb the toxic acidic material from the liquid intothe amino functional core particle, leaving the neutral porous layeressentially free of the toxic acidic material. The resultantnanoparticle with adsorbed toxic acidic material may be safe to handle,since the outer layer of the nanoparticle would be free of the toxicacidic material.

More generally, the layered nanoparticle may comprise (in the coreand/or in one or more layers) chelating and/or complexing groups thatare capable of interacting specifically with a particular chemicalspecies or class of chemical species in order to provide selectivesorption of that chemical species or class of chemical species. Thechelating and/or complexing groups may be either in the form of anon-releasable dopant or in the form of the material of the layeritself, or both. Such layered nanoparticles may be used for example forsorption of toxic chemicals, such as heavy metals. Thus a layerednanoparticle having a non-releasable dopant comprising a group capableof chelating lead (e.g. a bound EDTA group) may be used for removinglead from a solution.

The mean nanoparticle bulk density may be in the range 0.075 g/cm³ to2.2 g/cm³, or about 0.15 to 1.5, 0.18 to 1.0, 0.5 to 1.0, 0.5 to 0.75 or0.25 to 0.5 g/cm³, and may be about 0.075, 0.1, 0.15, 0.2, 0.25, 0.3,0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95,1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 or 2.2 g/cm³.

The nanoparticulate substance may be monodispersed, or may have a narrowparticle size distribution. The particle size distribution may be suchthat more than 50% of particles lie within 10% of the mean particlesize, or more than 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10%, and may besuch that about 10, 20, 30, 40, 50, 60, 70 or 80% of particles liewithin 10% of the mean particle size.

The nanoparticulate substance may have a mean particle diameter betweenabout 10 and 500 nm, or between about 10 and 100, 50 and 500, 50 and300, 50 and 100, 100 and 500, 250 and 500, 100 and 300 or 200 and 300nm, and may have a mean particle diameter of about 10, 20, 30, 40, 50,100, 150, 200, 250, 300, 350, 400, 450 or 500 nm, or may have a largermean particle diameter. The particles of the nanoparticulate substancemay comprise a core and between 1 and 10 layers, or layers, at leastpartially surrounding the core. There may be between 1 and 8, 1 and 5, 1and 3, 2 and 10, 5 and 10, 2 and 8 or 2 and 5 layers, and may be 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 layers, or may be more than 10 layers. Themean diameter of the core may be between about 5 and 500 nm, or betweenabout 5 and 150, 100 and 500, 100 and 500, 100 and 400, 100 and 300, 100and 200, 100 and 150, 5 and 125, 5 and 100, 5 and 80, 5 and, 50, 5 and30, 10 and 100, 30 and 100, 50 and 100, 100 and 150, 100 and 130, 130and 150, 10 and 80, 20 and 70 or 30 and 70 nm, and may be about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450 or 500 nm. Themean thickness of each layer or layer may, independently, be betweenabout 1 and 50 nm, or between about 1 and 30, 1 and 20, 1 and 10, 5 and30, 5 and 10, 10 and 50, 10 and 40, 10 and 30, 10 and 20, 20 and 50, 30and 50, 40 and 50, 20 and 40 or 20 and 30 nm, and may be about 1, 2, 3,4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nm. If more than one layer orshell is present, they may be different thicknesses or the samethickness. The core and/or the layers may be porous and may bemicroporous, or mesoporous, and may have a mean pore size of betweenabout 0.5 and 20 nm, or between about 0.5 and 10, 0.5 and 5, 0.5 and 4,0.5 and 3, 0.5 and 2, 0.5 and 1.7, 0.5 and 1, 1 and 5, 2 and 5, 3 and 5,4 and 5, 5 and 10, 7 and 10, 10 and 20, 5 and 15, 5 and 7, 1 and 3 or 1and 2 nm, and may have a pore size about 0.5, 0.6, 0.7, 0.8, 0.9, 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nm. Thus, forexample, both the core and all layers may be porous, or the core may benon-porous and the layers may be porous, or the core and an inner layermay be non-porous, and an outer layer may be porous. The core particleand the or each layer may, independently, have a porosity of betweenabout 0 and 80%, or between about 0 and 70, 0 and 60, 0 and 50, 0 and40, 0 and 30, 0 and 20, 0 and 10, 2 and 80, 10 and 80, 30 and 80, 50 and80, 10 and 50, 30 and 50, 20 and 60 or 20 and 40%, and may have aporosity about 0, 10, 20, 30, 40, 50, 60, 70 or 80%. The dopant mayrepresent between about 0 and 100 wt % of the core (i.e. the core mayhave no dopant or may be an active material which may or may not also bea dopant in one or more layers: the core may for example comprise amicronised drug particle or marker or quantum dot, as in the example ofthe In₂O₃ nanocrystals). The dopant may represent between about 0 and50, 0 and 25, 0 and 10, 0 and 5, 10 and 100, 50 and 100, 5 and 95, 10and 50 or 25 and 50 wt % of the core, and may represent about 0, 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 96, 97, 98, 99 or 100 wt % or the core The dopant may representbetween about 0 and 25 wt % of any particular layer surrounding thecore, or between about 0 and 20, 0 and 15, 0 and 10, 0 and 5, 5 and 25,10 and 25, 15 and 25, 20 and 25, 5 and 20 or 10 and 20 wt %, and mayrepresent about 0, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 wt % of any particularlayer surrounding the core.

The step of at least partially separating the layered nanoparticle(s)may comprise filtering, microfiltering, centrifuging, ultracentrifuging,settling, decanting or a combination of these. It may also comprise thestep of destabilising the suspension, and the destabilising may beperformed before said filtering, microfiltering, centrifuging,ultracentrifuging or settling.

The step of destabilising may comprise adding to the suspension adestabilising liquid. The destabilising liquid may be polar, and may bemiscible with the non-polar liquid. The destabilising liquid may bemiscible with water. It may be for example acetone, ethanol, methanol orsome other liquid. The step of destabilising may comprise changingtemperature, for example to a temperature at which the suspension is notstable. Depending on the phase diagram, the changing may be heating ormay be cooling.

The steps of washing may comprise contacting the layered nanoparticle(s)with a washing liquid (either aqueous or organic) and separating thelayered nanoparticle(s) from the washing liquid. For example any or allof the steps of washing may comprise suspending the layerednanoparticle(s) in the washing liquid, optionally agitating the combinedwashing liquid and the layered nanoparticle(s), and separating thelayered nanoparticle(s) from the washing liquid, for example using anyof the separation methods described above. Alternatively any or all ofthe steps of washing may comprise passing the washing liquid past and/orthrough the layered nanoparticle(s), which may be retained, for examplein a filter. The washing may be performed inside a decantation funnel byphase separation. The aqueous washing liquid may be water or an aqueousliquid, for example a salt solution. The organic washing liquid may be asolvent, and may be a polar or a non-polar solvent, for examplemethanol, ethanol, isopropanol, acetone, dichloromethane, chloroform,ethyl acetate, toluene or some other solvent, and may be a mixture ofsolvents. The step of washing may also comprise heating or cooling thesuspension to between about 10 and 70° C., or between about 10 and 50,10 and 30, 10 and 20, 20 and 70, 50 and 70, 20 and 50 or 30 and 50° C.,and may comprise heating or cooling the suspension to about 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65 or 70° C. The heating or cooling maybe to a temperature which prevents the system from forming a singlestable phase by changing the phase diagram of the system. The system mayalso be prevented from forming a single stable phase by using a mixtureof solvents to change the phase diagram.

The step of drying may comprise heating the layered nanoparticle(s). Theheating may be to a temperature below the temperature at which thedopant(s), if present, decomposes or deteriorates, and may be forexample between about 30 and 80° C., or between about 30 and 60, 30 and40, 40 and 80, 60 and 80 or 40 and 60° C., and may be about 30, 35, 40,45, 50, 55, 60, 65, 70, 75 or 80° C. Alternatively or additionally thestep of drying may comprise freeze-drying, for example as described inW01/62332 (Barbé and Bartlett, “Controlled Release Ceramic Particles,Compositions thereof, Processes of Preparation and Methods of Use”). Thestep of drying may additionally or alternatively comprise passing astream of gas over and/or through the layered nanoparticle(s). The gasmay be a gas that is inert to the layered nanoparticle(s) and to anydopant therein and/or thereon, and may be for example air, nitrogen,argon, helium, carbon dioxide or a mixture of these, and may be dried.The step of drying may additionally or alternatively comprise applying apartial vacuum to the layered nanoparticle(s). The partial vacuum mayhave an absolute pressure of for example between about 0.01 and 0.5atmospheres, or between about 0.01 and 0.1, 0.01 and 0.05, 0.1 and 0.5,0.25 and 0.5, 0.05 and 0.1 or 0.1 and 0.25 atmospheres, and may have anabsolute pressure of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4 or 0.5atmospheres.

In an embodiment of the process of the invention the step of providing asuspension comprising a core particle in a non-polar liquid comprisesthe steps of:

-   -   providing an emulsion comprising aqueous droplets dispersed in a        non-polar liquid, wherein the droplets comprise a catalyst for        hydrolysis of a first hydrolysable species, said first        hydrolysable species being capable of producing a first        condensable species upon hydrolysis; and    -   adding the first hydrolysable species to the emulsion so that        the first hydrolysable species hydrolyses within the droplets to        form a suspension of core particles in the non-polar liquid.

The aqueous droplets may be between about 5 and 150 nm in diameter, orbetween about 5 and 125, 5 and 100, 100 and 130, 120 and 150, 5 and 80,5 and, 50, 5 and 30, 10 and 100, 30 and 100, 50 and 100, 10 and 80, 20and 70 or 30 and 70 nm, and may be about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140 or150 nm in diameter.

The step of providing an emulsion may comprise the steps of:

-   -   providing a surfactant solution comprising a surfactant and a        non-polar liquid, and optionally a co-surfactant;    -   adding to the surfactant solution an aqueous solution comprising        a catalyst for condensation of a first condensable species and,        optionally, also comprising a first dopant;    -   forming an emulsion from the surfactant solution and the aqueous        solution;

The surfactant may be an anionic, cationic, non-ionic or zwitterionicsurfactant, and may be a monomeric or polymeric surfactant. Suitablesurfactants include nonylphenoxypolyethoxyethanol,C₉H₁₉C₆H₄(OCH₂CH₂)_(n)OH (NP series), or octylphenoxypolyethoxyethanolC₈H₁₇C₆H₄(OCH₂CH₂)_(n)OH (Triton series), where n is between 4 and 15,or between 4 and 9 or 9 and 15 or 7 and 12, and may be 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or 15. Suitable cosurfactants include C5 to C10n-alcohols (1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 1-nonanol,1-decanol). The concentration of the surfactant in the surfactantsolution may be sufficient to form a stable microemulsion in combinationwith suitable amounts of water and cosurfactant, and may be betweenabout 0.05 and 1M, or between about 0.05 and 0.5, 0.05 and 0.2, 0.05 and0.1, 0.1 and 1, 0.5 and 1, 0.1 and 0.5 or 0.1 and 0.2M, and may be about0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9 or 1M, or may be some other suitable concentration. Theconcentration of the cosurfactant in the surfactant solution may bebetween about 0 and 1M, or between about 0.05 and 1, 0.05 and 0.5, 0.05and 0.2, 0.05 and 0.1, 0.1 and 1, 0.5 and 1, 0.1 and 0.5 or 0.1 and0.2M, and may be about 0, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2,0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1M. The molar ratio ofcosurfactant to surfactant may be between 0 and 10 or between about 0and 5, 0 and 2, 0 and 1.5, 0 and 1, 0 and 0.5, 0 and 0.2, 0 and 0.1, 0.5and 2, 1 and 2, 1.5 and 2, 0.5 and 1.5 or 0.8 and 1.2, 0.5 and 10, 1 and10, 2 and 10, 5 and 10, 0.5 and 5 or 1 and 5 and may be about 0. 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10. The ratio ofwater in the aqueous liquid to the surfactant may be between about 2:1and 10:1, or between about 2:1 and 5:1, 3:1 and 10:1, 4:1 and 10:1, 5:1and 10:1, 3:1 and 8:1 or 4:1 and 6:1, and may be about 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1 or 10:1 on a w/w, w/v, v/v or molar basis.

Synthesis of Nanoparticles

FIG. 3 summarizes a process according to the present invention. Theprocess may be described as follows:

-   -   1. A surfactant solution is formed by dissolving a surfactant        and a co-surfactant in an non-polar solvent such as cyclohexane.    -   2. An aqueous solution is formed by mixing the active molecule        (dopant) to be encapsulated (e.g. dye or drug) with a catalyst        for the sol-gel reaction (e.g. base).    -   3. A stable microemulsion is then prepared by mixing the        surfactant and the aqueous solution as produced above. An        alkoxide precursor (e.g. TEOS) is then added to the        microemulsion and subsequently slowly diffuses to the water        pools (i.e. core of the emulsion droplets) where it is        hydrolysed.    -   4. After hydrolysis and condensation of the metal alkoxide for        at least 48 hours, the reaction temperature is increased to        55±5° C. for one hour to ensure completion of the reaction. This        stage results in the production of the core (or seed) particles.    -   5. More aqueous solution (which may contain another active        molecule) is then added. The mixture is stirred at 55±5° C. for        six hours to allow the added water to be transferred at the        surface of the core particles. This is important in order to        prevent empty micelle formation, which may be a source of        secondary nucleation. A surfactant solution containing        surfactant, co-surfactant and non-polar solvent is then added.        After the system is cooled to room temperature, further        precursor (e.g. TEOS) is added to grow the particles by forming        a layer around the original core particles. Synthesis parameters        such as water to surfactant molar ratio, surfactant        concentration are kept constant during these steps.    -   6. Steps 4 and 5 are repeated one or more times to produce the        required number of layers in the layered nanoparticles. Note        that by using this process, different dopants may be        incorporated in different locations (i.e. layers) of the layered        nanoparticles.    -   7. After the layered nanoparticles are formed, a polar solvent        (e.g. acetone) is added to the mixture to destabilise the        emulsion system and to extract the particles by settlement or        centrifugation. If required, more solvent is used to remove the        residual surfactant.    -   8. A salt solution is added and the particles are then further        washed with chloroform, collected in separation funnel, and        freeze-dried using a procedure described in W01/62332 (Barbé and        Bartlett, “Controlled Release Ceramic Particles, Compositions        thereof, Processes of Preparation and Methods of Use”). The        particles may also be processed in suspension. A polar solvent        (e.g. acetone) is added to the mixture to destabilise the        emulsion system and the particles are extracted by settling or        centrifugation. If required, more solvent can be used to further        remove the surfactant residues.

Factors that control the final particle size and size distributioninclude emulsion parameters (e.g. surfactant concentration,surfactant/water ratio, precursor/water ratio), the nature and amount ofmetal precursor, and the cycle time of step 4 and 5. The number oflayers depends on how many times steps 4 and 5 are repeated. The typeand concentrations of active molecules (dopants) may be altered at eachcycle. The location of each active molecule depends on when it isintroduced in the process, and the thickness of each layer may becontrolled by the amount of precursor added.

Comparison with Other Growth Method Reported in the Literature

In contrast to the experiments reported in the literature, a processaccording to the present invention uses a seed growth technique toproduce core-shell layered nanoparticles by maintaining the sameoil-water-surfactant (optionally with cosurfactant) phase diagram. Thismay be achieved by adding fresh W/O microemulsion, or fresh surfactantand non-polar solvent, to the reaction system during the growth of eachlayer. Thus, addition of a second non-polar solvent should not alter theoil-water-surfactant phase diagram significantly, and the secondnon-polar solvent should be miscible with the non-polar solvent whichwas used initially. The process may proceed continuously withoutextracting seeds. Another unique feature of the present invention is thepossibility of introducing new active molecules during each addition offresh microemulsion. Hence a novel approach can be developed to formcore-shell layered nanoparticles, by which more than one dopant can beencapsulated in the core or selected shell(s) (layer(s)). Theencapsulated molecules may then be released in a predetermined releasesequence depending on their location inside the multi-layered structure.This ability to control the payload in each layer enables the design ofdelayed (empty layer+loaded core), as well as pulsed (alternate emptyand filled layer) and sequential release (layers filled with differentpayload) systems. Furthermore, not only the overall particle size becontrolled, but the thickness of the core and each layer can betailored, thus potentially controlling the length of the release cycles.Although the encapsulation of multi-active molecules in particles orfilm has not been extensively studied, the inventors believe that thisadvanced controlled release technology may find applications not only intraditional applications for controlled release systems such as food,chemical, biocide, pesticide, pharmaceutical and cosmetic, but also inother areas such as optical sensor, biosensor, encryption andinformation technology.

Examples Materials

NP-9 [nonylphenoxypolyethoxyethanol, C₉H₁₉C₆H₄(OCH₂CH₂)_(n)OH, n=9](Fluka: MW 630, HLB 13.0) was used as received (i.e. contains less than0.08 wt. % of water as determined by Karl Fisher titration).Tetraethylorthosilicate (TEOS) (98%) from Sigma-Aldrich was also used asreceived. Organic solvents (cyclohexane, 1-pentanol, acetone,chloroform) were ACS spectrophotometric grade (99+%) (Sigma-Aldrich).All other chemical reagents used were A.R. grade. High purity Milli-Qwater (Millipore) was used for the preparation of all aqueous solutions;its resistivity was above 18.2 MΩ cm. All samples were stored in thedark.

Preparation of Encapsulant (Dopant) Solution:

Preparation of Copper (II) Tetramine Nitrate:

Copper (II) tetramine nitrate solution was prepared by adding 25 wt. %ammonia dropwise to a known quantity of copper (II) nitrate-hydrate,until a dark blue solution was formed. The solution was transferred to100 mL volume flask and the volume was adjusted to 100 ml by addingdiluted 25 volume percent of concentrated ammonia (i.e. 25 wt % NH₃).The prepared solution contained copper 16.69 mg/mL and its pH wasmeasured as 11.86.

Preparation of Cobalt (III) Hexamine Nitrate:

Cobalt (III) hexamine nitrate solution was prepared by similar procedureas described above for copper (II) tetramine nitrate solution, exceptthat during dissolution of cobalt nitrate-hydrate by concentratedammonia (25 wt. % NH₃), the solution was heated to boil for twentyminutes leading to formation of soluble cobalt (III) hexamine complexes.The final solution contained cobalt 16.69 mg/mL, and pH was 11.82.

Preparation of Rubpy:

A Rubpy [(tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate]solution was prepared by dissolving a fixed amount of ruthenium complexin a fixed volume of diluted ammonia (10 vol % of concentrated 25 wt %NH₃) to make a solution with a concentration of 2.5 mg/mL of Rubpy.

Preparation of CuPC:

A CuPC [copper(II) phthalocyanine-tetrasulfonic acid tetrasodium salt]solution was prepared by dissolving a fixed amount of copper complexinto a fixed volume of diluted ammonia (10 vol % of concentrated 25 wt %NH₃) to make a solution with a concentration of 5 mg/mL of CuPC.

Preparation of FITC:

A fluorescein isothiocyanate solution was prepared by dissolving a fixedamount of fluorescein into a fixed volume of diluted ammonia (10 vol %of concentrated 25 wt. % NH₃) to make a solution with a concentration of5 mg/mL of Fluorescein.

Preparation of In-DTPA or Ga-DTPA Solution:

A known amount of InCl₃ or GaCl₃ was weighed in 100 mL beaker, 10 gMilli-Q water was added to dissolve the salt. A fixed amount ofdiethylenetriamine pentaacetic acid (DTPA) was added so that the DTPA tometal element molar ratio was kept at 1.1:1. Concentrated ammonia (25 wt% NH₃) was added dropwise as the mixture was stirred magnetically. Afterall of the DTPA was dissolved, the metal-DTPA chelate was formed withformula as [Me-DTPA]²⁻ and structure as following:

The concentrated ammonia was added continuously until the pH of solutionwas above 11.50 and total volume was just below 100 mL. The solution wastransferred to 100 mL volume flask quantitatively, and water was addedto a final volume of 100 mL.Typical Preparation for Producing 50 Nm Silica Core

7.56 g of NP-9 (12 mmol) was dissolved in 60 mL cyclohexane in a 250 mLscrew capped container. 1.304 mL (12 mmol) of 1-pentanol was then added.This mixture was shaken vigorously for about 1 minute to prepare thesurfactant solution. Then 1.333 mol/L of ammonia solution (NH₄OH(pH˜11.86) 1.296 mL (i.e. 72 mmol of water) containing the activemolecules, such as a dye, was added. The mixture was stirred vigorouslyfor about thirty minutes to produce the microemulsion system. 0.546 mLof TEOS (2.4 mmol) was added into this magnetically stirredmicroemulsion, which was then left to stir for a fixed time (48-72hours) at room temperature, 22±2° C. This was followed by the additionof 50 mL of dry acetone to destabilise the microemulsion. The system wasfurther stirred vigorously for about 10 minutes. The silica particlesusually flocculated and could be separated by decantation; if nosignificant sedimentation was observed, the mixture was centrifuged at4000 rpm for 10 minutes. After sedimentation or centrifugation, theparticles were separated from the organic phase and washed three timeswith 50 mL of dry acetone (each time) to remove the surfactant. Afterdecantation of the acetone, about 20 mL NaCl aqueous solution was mixedwith particles and introduced in a decantation funnel. The resultingsuspension was further washed 6 times using chloroform (50 mL eachtimes) to remove the residual surfactant, ultrasonicated and freezedried using a Flexi-dry-84D freeze-drier (FTS Systems, Inc., StoneRidge, N.Y.). Using this procedure, 144 mg silica were produced in theformed of nanoparticles homogeneously distributed inside a NaCl matrix.TEM shows the particle size was in the range of 55±5 nm (FIG. 4 and FIG.5-A). The sodium chloride is used as a protective matrix to prevent thenanoparticles from aggregating and the weight ratio between silica andsodium chloride is kept around 15%.

Typical Experimental Procedures for Preparing, 100 Nm, 150 Nm and 200 NmSilica Nanoparticles

Preparation 2: 100 Nm (Core+1 Shell):

-   -   Produce 50 nm seeds using the procedure described above;    -   Instead of adding acetone to destabilised the microemulsion,        increase temperature to 55±5° C. and stir for one hour;    -   Then add 2.952 mL of 1.333 mol/L NH₄OH (pH˜11.86) (i.e.        equivalent water 144 mmol) and stir for 6 hours at 55±5° C.,        then cool down to room temperature;    -   Add 15.12 g NP-9 (24 mmol) mixed with 2.608 mL 1-pentanol (24        mmol), and 120 mL of cyclohexane and stir for 20 minutes;    -   Add 2.184 mL TEOS (9.6 mmol) to the mixture;    -   Stir 48-72 hours;    -   Wash, extract and dry the particles as described in Typical        preparation for producing 50 nm silica core above;        This procedure produces 720 mg of monodisperse 100 nm silica        nanoparticles. The corresponding TEM micrographs are presented        in FIG. 5-B.

Preparation 3: 150 Nm (Core+2 Shells):

-   -   Prepare 100 nm particles as describe above;    -   Instead of adding acetone to destabilised microemulsion, split        the emulsion into two aliquots;    -   Take one aliquot, increase its temperature to 55±5° C. and stir        for one hour. Then add 2.592 mL of 1.333 mol/L NH₄OH (pH˜11.86)        (i.e. equivalent water 144 mmol) and stir for 6 hours at 55±5°        C.;    -   Add 15.12 g of NP-9 (24 mmol) mixed with 2.608 mL 1-pentanol (24        mmol), and 120 mL of cyclohexane and stir for 20 minutes;    -   Add 2.184 mL TEOS (9.6 mmol) into the mixture;    -   Stir for 48-72 hours;    -   Wash, extract and dry the particles as described in Typical        preparation for producing 50 nm silica core above;        This procedure produces 936 mg of relatively monodisperse 150 nm        silica nanoparticles. The corresponding TEM micrographs are        presented in FIG. 5-C.

Preparation 4: 200 Nm (Core+3 Shells):

-   -   Prepare 150 nm particles as described above;    -   Instead of adding acetone to destabilised microemulsion, split        emulsion into two aliquots;    -   Take one aliquot and heat it up to 55±5° C., stir for one hour,        and add 2.592 mL of NH₄OH 1.333 mol/L (pH˜11.86) (i.e.        equivalent water=144 mmol); stir for an additional 6 hours at        55±5° C.;    -   Add 15.12 g NP-9 (24 mmol), mixed with 2.608 mL 1-pentanol (24        mmol) in 120 mL cyclohexane and stir for 20 minutes;    -   Add 2.184 mL TEOS (9.6 mmol) to the system;    -   Stir for 48-72 hours;    -   Wash, extract and dry particles as described in Typical        preparation for producing 50 nm silica core above;        This procedure produces 1044 mg of relatively monodisperse 200        nm silica nanoparticles. The corresponding TEM micrographs are        presented in FIG. 5-D.        Particle Characterisation

The size and morphology of silica particles were monitored using atransmission electron microscope (JEOL 2000 FXII or JEOL 2010F).

To monitor the amount of residual surfactant TGA/DTA experiments wereperformed using a Setaram TGA 24. Undoped samples were heated to 900° C.at a rate of 10° C./min. A typical diagram is shown in FIG. 6. Twodifferent decomposition reactions were observed by DTA: an endothermicreaction at about 100-200° C. associated with the evaporation of waterand other volatile components and an exothermic reaction atapproximately 400° C. associated with the combustion of the surfactant.The amount of bound surfactant was calculated as the difference betweenthe weight loss at 450° C. and the weight loss at 250° C. The surfactantresidue associated with the silica particles after washing five times inchloroform is about 3.0 wt % of the silica dry weight. This correspondsto about 1.65 molecules of NP-9 per 50 nm particle.

Release Rate of Dyes

After separation of the silica particles from the aqueous phase bycentrifugation, the particles were resuspended in 20 mL of simulatedbody fluid (SBF, pH 7.4/25° C.). The composition of SBF can be seen fromP. Kortesuo; M. Ahola; S. Karlsson; I. Kangasniemi; A. Yli-Urpo; J.Kiesvaara, “Silica xerogel as an implantable carrier for controlled drugdelivery—evaluation of drug distribution and tissue effects afterimplanation”, Biomater., 21, 193-198 (2000). The release was measured bycentrifuging at 3000 rpm for half an hour the suspension and measuringthe concentration of dopant released in the supernatant using UV-visiblespectroscopy. During the release study, the samples were store in 37° C.water bath, and kept in the dark.

Results and Discussion

Influence of the Emulsion Parameters on the Seed Size

The following three systems show the influence of the water to alkoxideratio on the size of the core. The corresponding TEM micrographs arepresented in FIG. 7.

-   -   System 1: NP-5: 0.2 mol/L; cyclohexane 50 mL; catalyst: 1.333        mol/L NH₄OH (pH˜11.86); TMOS: 3 mmol; [H₂O]/[TMOS]=20,        [H₂O]/[NP-5]=6; ageing 24 hrs. Particle diameter: 7.6 nm.    -   System 2: NP-9: 0.2 mol/L; cyclohexane 30 mL, catalyst: 1.333        mol/L NH₄OH (pH˜11.86); [NP-9]/[1-pentanol]=1, TMOS: 1.2 mmol;        [H₂O]/[TMOS]=30, [H₂O]/[NP-9]=6; ageing 24 hrs. Particle        diameter: ˜20 nm.    -   System 3: Triton X-100: 23.9 wt. %; Toluene: 71.4 wt. %; Water:        4.7 wt. % (5 g); catalyst: 1.333 mol/L NH₄OH; TMOS: 8 mmol,        [H₂O]/[TMOS]=34.7. Particle diameter: ˜25 nm.        Particle Growth by Sequential Addition of Precursors (without        Extra Emulsion)        Two experiments were conducted using TMOS and TEOS as silicon        precursors.

Experiment 1

-   -   NP-9: 0.2 mol/L; cyclohexane 50 mL; catalyst: 1.333 mol/L NH₄OH;        [NP-9]/[1-pentanol]=1, [H₂O]/[NP-9]=6. Particle size increases        gradually by sequentially adding extra silicon precursor, the        growth process is from FIG. 2. The results are displays in        series in FIG. 8.

Experiment 2

-   -   NP-9: 0.2 mol/L; cyclohexane 30 mL; catalyst: 1.333 mol/L NH₄O;        [NP-9]/[1-pentanol]=1, [H₂O]/[NP-9]=6. Instead of using TMOS,        TEOS is used as silicon precursor, TEM images are shown in FIG.        9.

Although it is clear, from the TEM micrographs, that TEOS provides asignificantly more important particle growth than TMOS, it is necessaryto consider the quantity added in both cases as well as the size of theinitial core. In the case of TMOS, the initial core was 22 nm and theaverage quantity added per cycle was 1.2 mmol to reach an averagediameter of 36 after 4 additions. This represent a growth of 3 nm percycle or 2.5 nm growth per mmol of TMOS added. In contrast, the initialcore for TEOS is 50 nm and the average diameter after 5 addition of atotal value of 15.6 mmol is 137 nm. This represents a growth of 5.6 nmper mmol of TEOS added. This difference in growth rate may be explainedby the difference in the number of nuclei (i.e. core particles) presentin both cases. If all the silicon alkoxide is converted into silica, foran equivalent amount of silica the number of the smaller cores observedin the TMOS synthesis should be larger than the number of the largercores observed in the TEOS synthesis. In fact the difference in coreparticle population should be inversely proportional to their size sincethe total volume of silica is constant. Furthermore, for an equivalentaddition of silicon alkoxide, if the number of core particles to becoated is twice as large, the increase in the core-shell size would beexpected to be approximately twice as small. Hence, the smaller growthobserved for the TMOS.

Importance of the Choice of the Microemulsion System

NP-9 was selected for producing microemulsion during the entire particlegrowing process. It is generally believed that increasing the alcoholconcentration leads to the gradual increase of the polydispersity ofparticles, as the microemulsion phase diagram is sensitive to thealcohol content. This limits the maximum concentration of alkoxideprecursor totally added since this precursor produces alcohol during itshydrolysis. The microemulsion with NP-9 as surfactant is less sensitiveto alcohol content, thus higher concentration of alkoxide precursor maybe utilised during the particle growth process. The presence of1-pentanol as a cosurfactant makes the reverse micelles more rigid, andthus changes the microemulsion from unstable to stable, and alsoimproves the uniformity of micelle size and subsequently the solidparticle size. In addition, NP-9 microemulsions permit higher[water]/[surfactant] values, thus enabling the production of largerparticles while maintaining the stability of microemulsion and theuniformity of particle size.

Particle Growth by Sequential Addition of Microemulsion and SiliconAlkoxide

Although it is possible to grow the particles by sequential addition ofalkoxide precursor, the particle growth is limited to some extent by thelimited supply of free water in the micelle core. In addition, in thismethod, the active molecules can only be loaded in core of theparticles.

The alternative process, described in the present specification,consists in providing an additional supply of water, in the form ofadditional micro-emulsion, to sustain the alkoxide hydrolysis andcondensation, and thus particle growth. A schematic representation ofthe process is provided in FIG. 10. Once the seed (core) particle isproduced, a fresh microemulsion is added to the suspension of seeds(core particles). During the mixing, newly added water droplets from thefresh microemulsion coalesce with the hydrophilic droplets containingthe seed particles, and the water adsorbs at the surface of the existingcore particles. Upon addition of alkoxide precursor, it reacts with thewater adsorbed at the particle surface, hydrolyses and then condenseswith the hydroxyl groups present at the particle surface. If thealkoxide concentration is kept below a certain level, the concentrationof silicon precursor remains below the supersaturation level and noextra nuclei are formed. The particles grow gradually by a classicalmonomer to nuclei addition. Examples of particle grown using this methodare presented in FIG. 5.

The addition of water, in the form of a micro-emulsion at each cycleenables the introduction of active molecule at different stages withdifferent concentration. This leads to the possibility of encapsulatingmore than one type of active molecule in different locations inside theceramic nano-particles. The species in outer shells can then be releasedearlier than the molecules located inside the core or inner shells. Sometypical TEM images of these core-shell structures are shown in FIG. 11.Precise composition analysis of each shell using EDX line profile couldnot be performed as the electron beam damages the sample and leads to aredistribution of the metals inside the particles (see FIG. 12).

Using this synthesis procedure, monodispersed ceramic particles may beproduced in the size range of 50-500 nm, commonly 10-300 nm. The finalsize of the ceramic particles depends on:

-   -   (a) microemulsion properties including the type of surfactant,        co-surfactant, solvent, water surfactant molar ratio, surfactant        to co-surfactant molar ratio and pH of water pool;    -   (b) type of alkoxide precursor and its concentration;    -   (c) number of cycles;    -   (d) amount of precursor added at each cycle; and    -   (e) other sol-gel reactions conditions such as the temperature,        pH etc.        Using the process of the present invention, layered        nanoparticles may be produced as discussed above to produce        silica particles in the presence of base (pH higher than 11).        Using appropriate selection of conditions and reagents the        synthesis condition may be shifted to neutral, weak acid, even        medium to strong acid environment, possibly using other ceramic        materials than silica.        Influence of Different Experimental Parameters on the Particles        Polydispersity        Influence of the Method of Addition

FIG. 13 shows the different ways of adding the fresh microemulsioncomposition. The seeds were prepared by using the following system (NP-93 mmol, 15 ml of cyclohexane, 1 pentanol 3 mmol, 0.324 ml of NH₄OH1.33M, water 18 mmol, TEOS 0.6 mmol aged for 48 h). Additionalmicro-emulsion (double the volume of initially used for the seeds) andTEOS were then added as follows:

-   -   Addition of the micro emulsion, stirring at 55° C. for 6 hours        and addition of TEOS followed by aging for 68 hours.    -   Addition of the surfactant, 1 pentanol and cyclohexane first        followed 10 minutes afterwards by the addition of the ammonia        solution. Stirring at 55° C. for 6 hours and addition of TEOS        followed by aging for 68 hours.    -   Addition of the ammonia solution first followed 10 minutes        afterwards by the addition of the surfactant, 1 pentanol and        cyclohexane. Stirring at 55° C. for 6 hours and addition of TEOS        followed by aging for 68 hours.    -   Addition of the ammonia solution first followed by stirring at        55° C. for 6 hours. Then addition of the surfactant, 1 pentanol        and cyclohexane followed by the addition of TEOS and aging of        the suspension for 68 hours.

All the different methods produced particles with the same size. In allsubsequent processes the last method was used.

Influence of the Number of Shells

To study the size limit on polydispersity, a suspension of 200 nmparticles (i.e. core+3 shells) was split into two aliquots. One aliquotwas heated to 55±5° C. while stirring for one hour. 2.592 mL of NH₄OH1.333 mol/L (pH˜11.86) (equivalent water 144 mmol) was then added andthe resulting mixture stirred for 6 hours at 55±5° C. 15.12 g of NP-9(24 mmol), 2.608 mL of 1-pentanol (24 mmol), and 120 mL of cyclohexanewere then added to the suspension. After stirring for 20 minutes, 2.184mL TEOS (9.6 mmol) was added to the system which was further stirred for48-72 hours. The particles were then washed, extracted and dried asdescribed in Typical preparation for producing 50 nm silica core above.The TEM micrographs of the resulting particles (i.e. core+4 shells) areshown in FIG. 14-a. Using the same procedure particles with an extrashell (i.e. core+5 shells) were produced (see FIG. 14-b). FIG. 14demonstrates that sequential growth beyond 200 nm leads to theproduction of polydispersed particles. FIG. 14-a reveals a bimodaldistribution with in addition to the targeted 230 nm particles a fewsmaller ones with a size around 150 nm. One more iteration led to apolydisperse size distribution ranging from 100 nm to 325 nm. Thisgradual appearance of smaller particles suggests the formation of newsilica nuclei resulting from incomplete coalescence of the addedmicro-emulsion with the droplets containing the particles. Moregenerally, the larger the nanoparticles, the broader the sizedistribution. A possible way to the preserve monodispersity of large(>250 nm) particles may be fractionation by sedimentation and thuselimination of second generation (smaller) nanoparticles.

Another practical limitation, facing the sequential growth of largeparticles is the time necessary to complete the whole cycle. Typicallyto build three shells (i.e. 200 nm diameter) it may take 8-10 days. Oneway to decrease the processing time may be to increase the reactiontemperature. Alternatively, using more hydrolysable alkoxide precursormay speed up the particle growth.

Influence of the Fresh Emulsion/Seed Emulsion Volume Ratio

Experimental results (FIGS. 15 a and b) show that dilution of the seedemulsion with fresh microemulsion does not always lead to uniformparticles. When the dilution (volume of fresh emulsion/volume of seedemulsion) is higher than 3, it produces polydispersed particles. Thissuggests that the amount of water that can be incorporated with existingparticles cannot beyond certain value. If more water is added then newwater-in-oil reverse micelle are formed and act as nucleation centresfor the formation of new silica particles, resulting in the productionof polydispersed particles.

Influence of the Mixing Method

FIG. 16 shows the importance of the method used for mixing the emulsioncontaining the seed particles and the fresh microemulsion. A fewdifferent ways can be used to induce the coalescence of the freshemulsion droplets with the existing droplets containing the seeds andthus ensuring the production of monodispersed particles. The firstapproach consists in shear mixing the resulting emulsion mixture at avery high shear rate (8000 rpm) to promote droplet collision andcoalescence. The second approach relies on increasing the collision rateof the droplets by increasing the temperature of the mixed emulsion. Itis important to note that, although theoretically full coalescence couldbe achieved at room temperature, the time required to achieve this makeit impracticable, as demonstrated by the polydispersed sample obtainedafter 18 h stirring at room temperature. Sonication (even for longperiod of time) does not induce droplet coalescence as demonstrated bythe bimodal distribution observed in FIG. 16-b.

Influence of the Silicon Precursor Reactivity

As discussed earlier, a less reactive silica precursor such as TEOSproduces bigger cores than more reactive precursor like TMOS. When TMOSis added in the same molar ratio as TEOS to the 50 nm pre-formed coreparticles, a bimodal distribution is obtained with 75 nm core-shellparticles and smaller 21 nm secondary particles (see FIG. 17). The 21 nmparticles correspond exactly to the core produced using TMOS (see FIG.7-b). This suggests the presence of a secondary nucleation stage, whichresults from the concentration of hydrolysed TMOS reaching rapidlysupersaturation and being relieved by secondary nucleation rather thancondensation with the existing cores. In contrast, for the less reactiveTEOS, the concentration of hydrolysed monomers rises more slowly andtherefore they can be consumed by reaction with the core surface beforetheir concentration reaches supersaturation and induces nucleation.

Organic Modified Silica (ORMOSIL) Core-Shell Structured Nanoparticles

Sol-gel synthesis in base leads to the production of mesoporousparticles (i.e. large pore size: about 4 nm), which generally exhibit arapid and uncontrolled release of their payload (typically organic dyeor small drug molecules <2 nm). In order to keep the encapsulatedmolecules inside the ceramic nanoparticles and avoid leaching during thewashing step, the ceramic matrix may be functionalised with active group(such as —NH₂, —SH, —COOH etc.), which will form a chemical bond withthe encapsulated molecules. Using such a strategy, a fluorescent dye(fluorescein isothiocyanate (FITC)) has been successfully encapsulatedin silica particles functionalised with amine groups (APTES). Therelease of the dye may then be triggered by screening or cleavage of theactive molecule-matrix interaction.

Layered nanoparticles were synthesised according to experimentalconditions described earlier. The ORMOSIL is either integrated in theparticle core or inside the second shell. The proportion of TEOS iseither 75 mol % and 25 mol %. The ORMOSIL precursor used in theseexperiments is aminopropyltriethoxysilane (APTES). The corresponding TEMimages are shown in FIG. 18 and the compositions of the ORMOSILcore-shell particles are displayed in Table 1.

TABLE 1 The composition of ORMOSIL core-shell particles Image ofDiameter FIG. 18 Core Shell-1 Shell-2 (nm) Image-a TEOS (100 mol. %)48-61 Image-b TEOS (75 mol. %) 43-65 APTES (25 mol. %) Image-c TEOS (75mol. %) TEOS (100 mol. %)  80-104 APTES (25 mol. %) Image-d TEOS (100mol. %) TEOS (100 mol. %) 85-90 Image-e TEOS (75 mol. %) TEOS (100 mol.%) TEOS (100 mol. %) 122-152 APTES (25 mol. %) Image-f TEOS (100 mol. %)TEOS (100 mol. %) TEOS (75 mol. %) 150-160 APTES (25 mol. %)Core-Shell Structured Nanoparticles Encapsulated with Two ActiveMolecules in Different Locations

Another advantage of the present process is the possibility ofencapsulating more than one dopant in the core particle or in any of theshells. Each molecule may be selectively encapsulated in differentlocations of the nanoparticles, and its concentration may also becontrolled at each growth cycle. Not only is it possible to control theoverall size by this process, but both the core size and the thicknessof each shell may be tailored, thus potentially controlling the lengthof the release cycles as well as the different release rates. Thefollowing examples are aimed at demonstrating the versatility of thistechnique as well as the flexibility and control of the encapsulationprocess.

Table 2 lists the composition of different samples with Cu(NH₃)₄ ²⁺ andRu(bPy)₃ ²⁺ doped in different location of the nanoparticles. Thecorresponding TEM images are presented in FIG. 19 and the photographs ofthe particles suspended in acetone are showed in FIG. 20. Table 3 liststhe composition of different samples with Cu(NH₃)₄ ²⁺ and Co(NH₃)₆ ³⁺doped in different location of the nanoparticles. The corresponding TEMimages are presented in FIG. 21 and the photographs of the particlessuspended in acetone are showed in FIG. 22. Table 4 lists thecomposition of different samples with CuPC²⁻ and Ru(bPy)₃ ²⁺ doped indifferent location of the nanoparticles. FIG. 23 shows the correspondingfreeze-dried powders (SiO₂/NaCl weight ratio: 15%) and the particlesresuspended in water.

TABLE 2 The components of product 1, Cu(NH₃)₄ ²⁺ and Ru(bPy)₃ ²⁺ asdopants Sample Core Shell-1 Shell-2 LNK-704 Cu: 10.82 mg Ru: 3.24 mgSiO₂: 72.00 mg SiO₂: 288.00 mg LNK-705 Ru: 1.62 mg Cu: 21.63 mg SiO₂:72.00 mg SiO₂: 288.00 mg LNK-706 Cu: 10.82 mg SiO₂: 288.00 mg SiO₂:72.00 mg LNK-707 Ru: 1.62 mg SiO₂: 288.00 mg SiO₂: 72.00 mg LNK-708 Cu:10.82 mg SiO₂: 288.00 mg Ru: 6.48 mg SiO₂: 72.00 mg SiO₂: 576.00 mgLNK-709 Ru: 1.62 mg SiO₂: 288.00 mg Cu: 43.26 mg SiO₂: 72.00 mg SiO₂:576.00 mg

TABLE 3 The components and particle size of product 2, Cu(NH₃)₄ ²⁺ andCo(NH₃)₆ ³⁺ as dopants. Sample Core Shell-1 Shell-2 Diameter LNK-753 Cu:10.82 mg 44-50 nm SiO₂: 72.00 mg Cu/SiO₂: 15 wt. % LNK-754 Cu: 10.82 mgCo: 21.63 mg 80-90 nm SiO₂: 72.00 mg SiO₂: 288.00 mg Cu/SiO₂: 15 wt. %Co/SiO₂: 7.5 wt. % LNK-755 Cu: 10.82 mg SiO₂: 288.00 mg 76-88 nm SiO₂:72.00 mg Cu/SiO₂: 15 wt. % LNK-756 Cu: 10.82 mg SiO₂: 288.00 mg Co:43.26 mg 102-132 nm  SiO₂: 72.00 mg SiO₂: 576.00 mg Cu/SiO₂: 15 wt. %Co/SiO₂: 7.5 wt. % LNK-757 Co: 10.82 mg 38-60 nm SiO₂: 72.00 mg Co/SiO₂:15 wt. % LNK-758 Co: 10.82 mg Cu: 21.63 mg 77-88 nm SiO₂: 72.00 mg SiO₂:288.00 mg Co/SiO₂: 15 wt. % Cu/SiO₂: 7.5 wt. % LNK-759 Co: 10.82 mgSiO₂: 288.00 mg 76-87 nm SiO₂: 72.00 mg Co/SiO₂: 15 wt. % LNK-760 Co:10.82 mg SiO₂: 288.00 mg Cu: 43.26 mg 104-135 nm  SiO₂: 72.00 mg SiO₂:576.00 mg Co/SiO₂: 15 wt. % Cu/SiO₂: 7.5 wt. %

TABLE 4 The components of product 3, CuPC²⁻ and Ru(bPy)3²⁺ as dopants.Sample Core Shell-1 Shell-2 LNK-819 Ru: 1.62 mg SiO₂: 288.00 mg SiO₂:72.00 mg LNK-820 CuPC: 3.24 mg SiO₂: 288.00 mg SiO₂: 72.00 mg LNK-821Ru: 1.62 mg SiO₂: 288.00 mg SiO₂: 576.00 mg SiO₂: 72.00 mg LNK-822 Ru:1.62 mg SiO₂: 288.00 mg CuPC: 12.96 mg SiO₂: 72.00 mg SiO₂: 576.00 mgLNK-823 CuPC: 3.24 mg SiO₂: 288.00 mg SiO₂: 576.00 mg SiO₂: 72.00 mgLNK-824 CuPC: 3.24 mg SiO₂: 288.00 mg Ru: 6.48 mg SiO₂: 72.00 mg SiO₂:576.00 mg

Although as mentioned before, elemental analysis and probing is notpossible due to electron beam damage, some clear core shell structurecan be evidence by TEM. The presence of an “onion” type structure (seeFIG. 21 b, d, e, f, g) confirms the possibility of selectively doping aspecific shell with a specific dopant. The colour changes observed inFIGS. 20, 22 and 23 with dopants located in different layer of thenanoparticles further illustrates the potential of this technology foroptical applications.

Encapsulation of Solid Core

An additional feature of the present invention is the ability toencapsulate solid particles inside a shell of metal oxide. The core maybe any species of powder such as metal oxide, semiconductor, quantumdot, magnetic particles or crystalline particles.

The shell(s) may comprise one metal oxide or a more than one differentmetal oxides, mixed metal oxides or metal oxide containing a dopant. Anexample of the preparation and encapsulation of crystalline In₂O₃nanocrystals inside silica shell is detailed below.

In₂O₃ nano crystals were prepared as follows: 1.017 g InCl₃ wasdissolved in 10 mL of pure water. 10 mL of NH₄OH 10M was added dropwiseto the solution while stirring and a precipitate was formed. Cl⁻ and NH₄⁺ ions were remove by successive washing (5-10 times with 50 mL of watereach time) until no smell of ammonia was detectable. 0.92 mL HNO₃ (1M)was then added to peptise the precipitate and the suspension stirredovernight at 55±5° C. A further 0.45 mL of HNO₃ 1 mol/L was added andthe suspension stirred again overnight at 55±5° C. The final pH wasmeasured at 2.284 and the TEM showed 50 nm*10 nm crystallites with welldefined facets.

The crystals were then coated using the following method. A solution Acontaining 6 mmol of NP-9, 6 mmol of 1-pentanol, 30 mL of cyclohexane,0.972 mL of NH₄OH 6.667 mol/L, was stirred until clear.

A solution B containing 6 mmol of NP-9, 6 mmol of 1-pentanol, 30 mL ofcyclohexane, 0.324 mL of the In₂O₃ nanocrystals in suspension wasstirred until clear.

The two solutions A and B were mixed together and 2.4 mmol of TEOS addedto the mixture. The solution was aged for 72 hrs. 50 ml of dry acetonewas then added to the resulting suspension to destabilise themicroemulsion and recover the particles. The particles were washedseveral times with acetone and examined by TEM (see FIG. 24). The TEMmicrographs clearly demonstrated the encapsulation of the 50 nm rodsinside silica nano-particlesEncapsulation of a Radioactive Tracer in the Core or Shell

The encapsulation of chelated species such as In-DTPA and Ga-DTPA in anyselected locations (core or selected shell) of silica particles has beensuccessfully achieved. The metal content as analysed by EDS wastypically 1-2 wt % of the silica. The particle size and sizedistribution were found to be independent of the presence or absence ofdopants. This confirms the ability to exercise independent control overparticle size and internal structure achieved by the process of thepresent invention.

Radioactive tracers such as ⁶⁷Ga-DTPA may also be easily doped insidesilica nanoparticles, thus producing particles that are traceable invivo. Such particles were prepared by the following procedure. ⁶⁷GaCl₃(t_(1/2): 3.261 days) was received from ANSTO-Cyclotron with activity 2GBq/mL in 0.1 mol/L HCl solution. The solution was diluted 1 in 10 withwater. 0.428 mL of concentrated ammonia (25 wt. % NH₃) containing DTPA3.9 mg/l was added to 0.22 mL ⁶⁷GaCl₃ solution at pH about 2.

The resulting solution may be used as the aqueous solution in theprocesses described earlier to produce particle with either activegallium in the core, in the outside shell or throughout the wholeparticle. These particles may be used in biodistribution studies toinvestigate the location of silica particles in rats using gammacounting as a detection method. Potential application of suchnanoparticles may be envisaged both in radiotherapy and radio-imaging.

Release from Core-Shell Particles

As mentioned previously, base catalysed sol-gel synthesis leads to theproduction of mesoporous particles (i.e. about 4 nm), which generallyexhibit a rapid and uncontrolled release of their payload. The notableexception to this is the encapsulation of organometallic complexes,which may exhibit some interaction between metal cation and silicasurface. In this case, the interaction of the surface with the complexensures encapsulation and temporary locking of the molecule inside thesilica nanoparticle structure. As the particles are suspended in aqueousphase, the strong affinity of the silica surface for water can graduallydisplaced the adsorption equilibrium with the active organometalliccomplex thus releasing the molecule out of the silica matrix. In mostcases the release is achieved by gradual dissolution of the silicamatrix.

To demonstrate the potential for sequential release, two sets ofnanoparticles with different dopants in the core and outer shell weresynthesized: a) sample LNK-822 with CuPC in the outer shell and Rubpy inthe core, and b) sample LNK-824, with Rubpy in the outer shell and CuPCin the core. The release from those two samples was monitored usingUV-vis spectrometry. A UV visible spectrum of the two dyes in water isshown in FIG. 25 and a spectrum of the two samples (LNK 822 and 824)after 32 days of leaching in SBF is shown in FIG. 26. The maximumabsorption of CuPC is at 556 nm, and at 390 nm for Rubpy. The overlapbetween the two peaks is small and can thus be neglected in a firstapproximation. No shift in the maximum absorption wavelength (for CuPCand Rubpy) with pH is observed in the pH range of 1-12 (corresponding tothe particle synthesis range). A red shift of approximately 60 nm isobserved for both dyes, from 390 nm to 450 nm for Rubpy and 556 nm to615 nm for CuPC when the media changes to SBF (see FIG. 26). In sampleLNK-824, a slight absorption can be seen in around 615 nm suggestingthere is slight amount of CuPC released as there is no absorption forRubpy at that region. However, due to the existence of slight absorptionof CuPC at 450 nm, it is hard to quantify the Rubpy release, especiallyfor such a small amount.

The corresponding release curves for the two core shell particles areshown in FIG. 27. When CuPC was encapsulated in the outer shell, it wasreleased gradually in 10 days. After that time, a constant concentrationof CuPC for a few days was observed suggesting that most of dye had beenreleased. A very small amount of the Rubpy from the core was alsoreleased although the exact amount is not easily quantifiable.Interestingly, the colour of LNK-822, which was green initially (seeFIG. 23), turned to orange (i.e. the colour of Rubpy dopednanoparticles) after release of CuPC from the outer shell, thusconfirming visually the sequential release of the CuPC. On the otherhand, when Rubpy was doped in the outer shell and CuPC was in core(LNK824) the release curve displays that in 5 days most of Rubpy wasleached. No detectable leaching of CuPC from core was observed. Duringthe leaching, the colour of particles changed from yellow to green/blue(the original colour of the particles containing only CuPC). Theseexperiments confirm the potential use of these multilayered particlesfor sequential release applications.

When no strong interaction between the molecule and the silica poresurface is present, the ceramic matrix may be functionalised with activegroups (such as —NH₂, —SH, —COOH etc.), which can form a chemical bondwith the encapsulated molecules. Using such a strategy, a fluorescentdye (fluorescein isothiocyanate (FITC)) has been encapsulated in silicaparticles functionalised with amine groups (APTES). The release couldthen be triggered by screening or cleavage of the active molecule-matrixinteraction. Another possibility to encapsulate small molecules insidethe mesoporous particles is to conjugate them with larger molecules suchas dextran or a dendrimer prior to encapsulation.

The present invention provides a process for manufacture layerednanoparticles, with core-shell structures and which contain one or moreactive molecules or dopants encapsulated in different locations (i.e.shells or layers) of the nanoparticles. Advantages of the technologyinclude the ability to control precisely the overall particle size, coresize, shell thickness, concentration of active species in each shell, aswell as the release sequence and release rate of each encapsulateddopant.

The inventors have demonstrated that small organometallic molecules canbe encapsulated inside the particles. These molecules include metalcomplex compound (e.g. copper tetramine, cobalt hexamine, indium-DTPA,gallium-DTPA), inorganic dye such as Rubpy[(tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate], CuPC [copper(II) phthalocyanine-tetrasulfonic acid tetrasodium salt], andfluorescence dye FITC (fluorescein isothiocyanate). Even complex objectssuch as In₂O₃ nanocrystals may be encapsulated. More importantly, theinventors have shown that it is possible to encapsulate more than onedopant at different locations inside the nanoparticles with differentloading value. The nanoparticle structure i.e. core size and layerthicknesses may be precisely controlled by the processing parameterssuch as the nature and quantity of precursor added. The monodispersityof the particles may be maintained throughout the growth process byensuring that all the water added at each cycle migrates to the surfaceof the existing particles. Residual empty water droplets (i.e. notcontaining a particle) may act as secondary nucleation centers. Althoughthe growth kinetics may be accelerated by increasing the amount ofprecursor, adding a catalyst or using less seed materials,polydispersity is introduced after the 4th cycle. Extreme dilution ofthe seed emulsion also leads to the production of polydispersednanoparticles.

Although the nanoparticles produce by the present process are suitablefor intravenous drug delivery, they may have application in informationtechnology for high-density optical memory storage, specific opticalsensors or biosensors, and in the protection/surface modification ofnanoparticles from corrosion/oxidation, sintering and coalescence,change of surface properties (surface, charge, zeta potential,suspensibility), etc. In the long term, the technology may be employedto develop nanoparticles comprising core particles containingencapsulated active molecules coated by shell(s) comprising organicmodified ceramic precursor exhibiting functional group like —NH₂, —SH,—COOH, which may be easily functionalised by antibodies or peptides foractive drug targeting application.

FIGS. 28a to 28f show diagrammatic representations of several differentlayered nanoparticles according to the present invention. It should benoted that these representations show only a few of the many differenttypes of nanoparticle which may be made according to the invention, andthat the particular methods for using the nanoparticles are by way ofexample only. Other types of particles and methods for using them arepossible and are envisaged in the present specification. Thus thedescriptions below are to be taken as examples only, and are not to betaken in any way as being limiting with respect to the scope of theinvention.

With reference to FIG. 28a , layered nanoparticle 10 comprises porouscore particle 15 comprising first releasable dopant A and a porous layer20 comprising second releasable dopant B surrounding core particle 15.When nanoparticle 10 is place in a suitable liquid environment,releasable dopant B is released first from nanoparticle 10, and thenreleasable dopant A is released. The period of time over which dopant Bis released may depend on the thickness of layer 20, and the period oftime over which dopant A is released may depend on the thickness oflayer 20 as well as the size of core particle 15. The release rates maybe dependent on the nature of the dopants as well as the nature and poresize of core particle 15 and layer 20. Alternatively, if dopant A is anon-releasable dye, nanoparticle 10 will appear coloured by dopant A,and will release releasable dopant B. As a further alternative, ifdopant B is a non-releasable dye and dopant A is a releasable dopant,then nanoparticle 10 will appear coloured by dopant B, and will releasedopant A.

With reference to FIG. 28b , layered nanoparticle 30 comprises porouscore particle 35 comprising releasable dopant A and porous layer 40comprising no releasable dopant. Porous layer 40 surrounds core particle35. When nanoparticle 30 is placed in a suitable liquid environment,releasable dopant A is released from nanoparticle 10 after a delay. Thelength of the delay depends on the thickness and porosity of porouslayer 40.

With reference to FIG. 28c , layered nanoparticle 50 comprises porouscore particle 55 comprising first releasable dopant A, first porouslayer 60 surrounding core particle 55 and second porous layer 65surrounding first porous layer 60. First layer 60 comprises noreleasable dopant and second porous layer 65 comprises second releasabledopant B. When nanoparticle 50 is placed in a suitable liquidenvironment, second releasable dopant B and first releasable dopant Aare released sequentially, with a delay between release of dopants B andA. The length of the delay is dependent on the thickness and porosity offirst layer 60.

With reference to FIG. 28d , layered nanoparticle 70 comprises porouscore particle 75 comprising first releasable dopant A, first porouslayer 80 comprising second releasable dopant B surrounding core particle75 and second porous layer 85 comprising third releasable dopant Csurrounding first layer 80. When nanoparticle 70 is place in a suitableliquid environment, releasable dopant C is released first fromnanoparticle 70, then releasable dopant B is released and finallyreleasable dopant A is released. The timing of the release of dopants A,B and C from nanoparticle 70 depend on the thicknesses and porosities oflayers 80 and 85 and the size of core particle 75.

With reference to FIG. 28e , nanoparticle 90 comprises porous coreparticle 95 with concentric porous layers 100, 105, 110 and 115surrounding core particle 95. Core particle 95 and concentric layers 105and 115 comprise releasable dopants A, B and C respectively, whereaslayers 100 and 110 contain no releasable dopant. When nanoparticle 90 isplace in a suitable liquid environment, releasable dopant C is releasedfirst from nanoparticle 70, then releasable dopant B is released andfinally releasable dopant A is released. There is a delay between therelease of dopant C and dopant B, and another delay between the releaseof dopant B and dopant A. The delays may be controlled by thethicknesses and porosities of layers 110 and 100 respectively.

With reference to FIG. 28f , nanoparticle 120 comprises porous coreparticle 125 comprising releasable dopant A, and porous layer 130surrounding core particle 125. Porous layer 130 comprises releasabledopants B and C. When nanoparticle 120 is place in a suitable liquidenvironment, dopants B and C are released first from nanoparticle 120,and then dopant A is released. Alternatively, if dopant A is anon-releasable dye, nanoparticle 120 will appear coloured by dopant A,and will release releasable dopants B and C. As a further alternative,if dopant B is a non-releasable dye and dopants A and C are a releasabledopants, then nanoparticle 120 will appear coloured by dopant B, andwill release dopants C and A sequentially.

Multi-Dye Encapsulation

In experiments described above, CuPC²⁻ and Ru(bPy)₃ ²⁺ have beenencapsulated in silica nanoparticles with CuPC²⁻ in core and Ru(bPy)₃ ²⁺in outer shell, and vice versa. The amount of each dye in core ofsample-I and outer shell of sample-II is different. Hence, these twosamples display different colours visually (FIG. 23, sample LNK-822 andLNK-824). In the following experiment, the same amount of each dye wasdoped in different samples, as shown in Table 5. The particles showidentical colour visually after synthesis and freeze-drying, suggestingthat the colour of the nanoparticles depends on the dye components andtheir concentration rather than their location in the nanoparticles.

TABLE 5 The component of dye doped nanoparticles. Sample Core Shell-1Shell-2 LNK-879 Ru: 3.24 mg SiO₂: 288.00 mg CuPC: 6.48 mg SiO₂: 72.00 mgSiO₂: 576.00 mg LNK-880 CuPC: 6.48 mg SiO₂: 288.00 mg Ru: 3.24 mg SiO₂:72.00 mg SiO₂: 576.00 mg

FIG. 29 shows the dye release of each sample in PBS (phosphate buffersolution) at 37° C. Most of the dye released by the multi-layeredparticles was that encapsulated in the outer shell i.e. CuPc andRu(bPy)₃ ²⁺ for LNK879 (FIG. 29-A) and LNK880 (FIG. 29-B) respectively.For LNK879 the concentration of CuPC remained roughly constant betweenday 18 and day 40. After that, it decreased substantially. The inventorshypothesise that this may have occurred due to decomposition of the CuPCor association of the CuPC with the walls of the container. In the earlystages of the release profile, the concentration of CuPC increased orremained substantially constant. The inventors hypothesise that this mayindicate that the dye release rate is higher than the rate ofdecomposition. FIG. 29-A shows that the dye (Ru(bPy)₃ ²⁺) in the corestarted to release after about 30 days, suggesting that sequentialrelease of the content of the multi-layer particles did occur. In FIG.29-B, the dye (Ru(bPy)₃ ²⁺) in shell showed continuous release up to 64days, while a slight amount of CuPC was leached simultaneously and thendecomposed after 50 days. It appears from these results that the releaserate profile may be adjustable depending upon the nature of differentdopants in different locations in the particles. FIG. 29 A shows thesequential release, with an initial release of dye from the shell forthe first two weeks and the release of another dye from the core after30 days. The decrease in intensity of the dye from the shell may beexplained by degradation with time of CuPc visible also from figure B(CuPc in the core). FIG. 29-B does not show sequential release but agradual release of the ruthenium dye from the shell and a small burstrelease from the core.

Formation of ORMOSIL Shell to Enhance the Encapsulation of Organic Dye

In experiments described earlier, the fluorescent dye fluoresceinisothiocyanate (FITC) was encapsulated inside a silica matrix inpresence of coupling agent such as APTES because of the chemical bondingbetween isothiocyanante (—N═C═S) group and amine group. In the presentexperiment an attempt was made to encapsulate the organic dye insidesilica nanoparticles coated with ORMOSIL shell(s). It was expected thatthis would narrow the pore size and thus decrease the dye release rate.The experimental flow chart is shown in FIG. 30 and the correspondingTEM images are displayed in FIG. 31. The synthesis followed the typicalprocedure, as described earlier. Several options were explored in orderto modify the particle surface. The components of each sample and thecorresponding particle size of final products are displayed in Table 6.Particles a represent the initial cores made with TEOS only. Particles bare the core made using a precursor mixture of 75% TEOS and 25% APTES.Particles c are core-shell particles made using TEOS only. Particles dand e have ORMOSIL modified shells, with 30 mol. % APTES for d and 50mol. % APTES for e. Particles f have ORMOSIL core and ORMOSIL shell. Allparticles had approximately the same particle size, however ORMOSILmodified particles showed slight broader particle size range.

The encapsulation efficiency of orange-II was measured by UV/Visabsorption after combining the organic phases during synthesis andwashing procedures. Orange II is an organic dye, and thus differssignificantly different from the previous example of RuBpy and CuPC inthat it is purely organic and does not have strong interaction with thesilica surface. The values of three samples (LNK 1120, LNK1121 andLNK1123) with either no APTES in the outer shell, or 30 mol % APTES inthe outer shell, averaged about 5-14 wt. %, however, the encapsulationefficiency of sample with 50 mol. % APTES in the outer shell (LNK1122)was about 45 wt. %. The encapsulation efficiency of organic dye appearsto depend, amongst other factors, on the nature of the dye, its amount,the dye to silica weight ratio, ORMOSIL mole percentage and thethickness of ORMOSIL containing layer. This indicates that the organicgroups of ORMOSIL precursor may play a role in preventing the dyeleaching from silica matrix during synthesis and washing. Furthermore,this effect appears to be dependent on the function group of eachORMOSIL precursor. It was found that GLYMO (epoxyfunctional silane) wasmore efficient than most ORMOSILs in preventing dyes from leaching outvery rapidly.

TABLE 6 The components and particle size of ORMOSIL shelled particles.LNK-1120 LNK-1121 LNK-1122 LNK-1123 Sample (FIG. 31-c) (FIG. 31-d) (FIG.31-e) (FIG. 31-f) Orange-II 2.592 mg 2.592 mg 2.592 mg 2.592 mg in coreCore TEOS: 2.4 mmol TEOS: 2.4 mmol TEOS: 2.4 mmol TEOS: 1.80 mmolprecursors APTES: 0.60 mmol Core size 50-60 nm 50-60 nm 50-60 nm 43-65nm Shell TEOS: 9.6 mmol TEOS: 6.72 mmol TEOS: 4.8 mmol TEOS: 6.72 mmolprecursors APTES: 2.88 mmol APTES: 4.8 mmol APTES: 2.88 mmol Particlesize 95-115 nm 86-126 nm 80-130 nm 84-122 nm Encapsulation 13.1 wt. %5.0 wt. % 44.8 wt. % 13.9 wt. % efficiencyOther Methods to Increase Particle Size while Maintaining ParticleMonodispersity High Surfactant Concentration

It was observed that when the particle size is above 100 nm, theemulsion system becomes cloudy, the particles start to precipitate outof the water droplets and finally form two physical phases withoutagitation. One could postulate that the co-surfactant loses its functionand that a higher concentration of surfactant might maintain particlesin suspension. FIG. 32 shows the TEM images of silica particles producedusing various surfactant conditions. The initial seeds (core-shellparticles with diameter between 74 and 86 nm as shown in FIG. 32, a andb) were produced using the typical synthesis procedure with 0.2 mol/LNP-9 and 0.2 mol/L 1-pentanol with [water]/[NP-9] mole ratio at 6.

The core+2-layers and core+3-layers particles were formed from the seedsproduced above. Without the addition of extra co-surfactant(1-pentanol), the surfactant concentration was increased to 0.4 mol/L([water]/[NP-9] mole ratio=3 then), and 0.6 mol/L ([water]/[NP-9] moleratio=2). FIGS. 32 c and e shows that the addition of the first shellwithout extra cosurfactant produces particles which are mono-dispersed.However, as another layer grows (FIGS. 32 d and f), smaller particleswith diameter around 20 nm are produced. These results suggest that thepresence of co-w surfactant is critical to the formation of monodisperseparticles even when the particle are larger than about 100 nm and startto settle.

When both surfactant and cosurfactant are increased from 0.2 mol/L to0.4 mol/L, the particles with two or three shells retain a narrow sizedistribution (FIGS. 32 g and h), but without a significantly largergrowth compared to those synthesised with 0.2 mol/L of surfactant.

Combination of Two Growth Processes

As discussed above, the particle size increases gradually with more andmore silicon precursor being introduced in the emulsion (FIG. 2).However, the addition of alkoxide only (no additional water) leads tothe production of particles with a core containing the active dopantssurrounded by an empty shell. Moreover, after the addition of a certainquantity of alkoxide, all the water in the water pool is consumed and nofurther growth is possible. This is further exacerbated inmicroemulsions because only a small percentage of the water present inthe water pool is free and able to participate in the condensation ofthe alkoxide. Most of the water is bound to the micellar wall assolvation water for the surfactant polar head.

In one form of practicing the present invention, more emulsion is addedand the additional water is transferred onto the existing particlesurface by incubation step at higher temperature (about 55° C.) forseveral hours (FIG. 3). Nevertheless, after several growth cycles, thesilica particles become polydispersed. This may be explained by the factthat during each incubation process, the water is not only distributedon existing particle surface, but it also forms new empty waterdroplets. Another factor to consider is that with an increase inparticle size, the emulsion system is no longer homogeneous andthermodynamically stable and the particles are capable of settling. As aresult, the number of incubation process should be kept to a minimum inorder to maintain the monodispersity, or as narrow as possiblepolydispersity.

FIG. 33 illustrates the process of production of layered particles bycombining the two growth techniques described above (i.e. multi-additionof TEOS interspersed with addition of emulsion components). During eachincubation cycle, the silicon precursor is added twice rather than oncein typical method. FIG. 34 displays the TEM images of particles at eachgrowth stage with water to surfactant mole ratio at 6 for the wholeprocess. The initial seeds were about 55 nm. After 3 incubation cyclesthe initial seeds were coated with 7 layers, with particle diameter inthe range of approximately 200-240 nm. At this point, the water tosilicon alkoxide mole ratio was slightly above 4. The results of thisnew method show that the particle size may be increased above 200 nmusing a smaller number of incubation steps.

One might argue that a higher water to surfactant molar ratio should beused for the whole growth procedure, thus providing more water to thesystem. FIG. 35 shows the TEM images of particles synthesised with awater to surfactant mole ratio at 9 for the growth process. Compared toa [water]/[surfactant]=6 system, the initial seeds are not larger for a[water]/[surfactant]=9 system. However, the particle size increased morequickly for the latter under the identical growth procedure. Forcore+3-layer particles, the size increased to around 158-172 nm but thesubsequent addition, small particles were generated, which indicatesthat monodispersed particles cannot be grown better using this highwater to surfactant ratio.

Synthesis at High Temperature

As noted previously, it may take several days to produce layerednanoparticles. In an attempt to shorten the reaction time experimentswere conducted at higher temperature. FIG. 36 shows the TEM images ofnanoparticles synthesised at 35° C. or 50° C. 35-50 nm particles wereproduced at 35° C. for a 20 hours ageing time. The particle sizeincreased to about 48-62 nm when an ageing time of 48 hours was used atthe same temperature. At 50° C., the particle size increased from 37-52nm to 48-70 nm as the ageing time was increased from 20 to 48 hours. Incomparison, at room temperature, the size range of nanoparticles wasaround 50-60 nm for 48 hour ageing (FIG. 34-a). The results indicatethat the particle size increases with time but is largely independent ofthe reaction temperature. High temperature reaction did not lead toquick completion of sol-gel reaction, but produced particles with abroader size distribution.

FIG. 37 shows core-shell particles produced at a higher reactiontemperature with a water to surfactant mole ratio at 8. The averageparticle size increased about 10-15 nm as the reaction time wasincreased from 20 to 48 hours, regardless of reaction temperature (35 or50° C.).

Incubation Effect on Particle Size

As mentioned earlier, to produce layered nanoparticles the seeds weremanufactured at room temperature followed by an incubation stage at55±5° C. for one hour to ensure that the majority of TEOS was completelyreacted. The new emulsion components were then added to the suspensionfollowed by an incubation of the mixture at 55±5° C. for 6 hours toallow all newly added water to adsorb on the existing particle surface.FIG. 38 shows the silica particle size at different incubation stages.Before incubation, the particles were in the size range of approximately76-88 nm. After incubation for one hour under 55±5° C., the particlesize increased slightly to about 82-96 nm. However, after adding extrawater and then incubating the system for 6 hours at 55±5° C., theaverage particle size was hardly changed, exhibiting slightly broadersize distribution. No particle size increase was observed for longerincubation times, suggesting that almost all silicon alkoxide wasreacted after one-hour.

The invention claimed is:
 1. A layered nanoparticle comprising aninorganic core particle or an organically modified inorganic coreparticle and one or more porous ceramic layers at least partiallysurrounding said core particle, wherein at least one region of thenanoparticle selected from the core and the one or more layers has adopant substantially homogeneously distributed therein, said layerednanoparticle being capable of releasing the dopant.
 2. The layerednanoparticle of claim 1 wherein: the core particle is porous and has acore dopant substantially homogeneously distributed therein; and atleast one of the layers is a porous layer having a layer dopantsubstantially homogeneously distributed therein, said porous layer atleast partially surrounding the core particle.
 3. The layerednanoparticle of claim 2 wherein the nanoparticle is capable of releasingthe layer dopant and the core dopant sequentially.
 4. The layerednanoparticle of claim 1, said nanoparticle being formed by forming alayer on the core particle.
 5. The layered nanoparticle of claim 1wherein the release rate of the dopant is dependent on the nature of thedopant and the nature and pore size of the core particle and the layer.6. A nanoparticulate substance comprising a plurality of layerednanoparticles according to claim 1, said dopant being an activesubstance, said nanoparticulate substance having a narrow particle sizedistribution.
 7. A method for delivering a nanoparticulate substance toa biological fluid comprising exposing a nanoparticulate substanceaccording to claim 6 to the biological fluid, said dopant being anactive substance and the biological fluid being capable of at leastpartially releasing the releasable substance from the layerednanoparticles.
 8. A method for administering a drug to a patient in needthereof, said method comprising delivering to the patient a compositioncomprising the nanoparticulate substance according to claim 6, whereinthe active substance is a drug, wherein at least one region of thenanoparticle selected from the core and the one or more layers is porousand comprises the drug substantially homogeneously distributed therein,and said nanoparticles being such that the drug is releasable therefrom.